2 Flavodoxins and Electron-Transferring Flavoproteins

Flawodoxins and Electron-Transferring Flavoproteins STEPHEN G. MAYHEW

MARTHA L. LUDWIG

I. Introduction. . . . . . . . . . . . . . . . 11. Flavodoxins . . . . . . . . . . . . . . . . A. Background . . . . . . . . . . . . . . . B. Structures . . . . . . . . . . . . . . . C. Flavin-Protein Interactions: Chemical and Physical Studies Solution . . . . . . . . . . . . . . . 1). Spectroscopic Properties . . . . . . . . . . . E. Oxidation-Reduction Potentials . . . . . . . . . F. Reactivity . . . . . . . . . . . . . . . 111. Electron-Transferring Flavoprotein . . . . . . . . . A. Introduction. . . . . . . . . . . . . . . B. Molecular Properties . . . . . . . . . . . . C. Catalytic Properties . . . . . . . . . . . .

. .

. .

in

. .

. . . . . .

57 58 68 66 82 88 98 102 109 109 111 116

1. Introduction

This chapter discusses two classes of flavoproteins which function solely to mediate electron transfer between the prosthetic groups of other proteins. Beinert ( 1 ) and co-workers discovered the first flavoprotein of this type, a soluble FAD protein obtained from mitochondria which couples the oxidation of acyl-CoA dehydrogenases to the reduction of components of the terminal electron transfer chain. According to its function the protein was termed “electron-transferring flavoprotein” (ETF). 1. H. Beinert, “The Enzymes,” 2nd ed., Vol. 7, p. 467, 1963. 57

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STEPHEN G. MAYHEW AND MARTHA L. LUDWIG

More recently, a different class of flavoprotein carriers has been isolated from microorganisms. Because of their functional interchangeability with the ferredoxins, these proteins have been called flavodoxins. The major portion of this chapter is devoted to the flavodoxins because they are the first flavoproteins for which three-dimensional structures have been determined. II. Flavodoxinr

A. BACKGROUND 1. Discovery, Nomenclature, and Distribution In the decade that followed the discovery of ferredoxin (2,S),a further class of microbial proteins which transfer electrons at low potential was recognized. These proteins are also small and acidic and in many reactions they substitute efficiently for ferredoxin. However, in contrast to the ferredoxins which contain iron and acid-labile sulfide, proteins in the second group utilize a molecule of flavin mononucleotide as their redoxactive component. Smillie (4,6) purified the first flavoprotein of this kind from extracts of the blue-green alga Anacystis niduhns and termed the protein “phytoflavin.” Shortly afterward, an FMN protein with similar catalytic properties was isolated from a strictly anaerobic bacterium, Clostridiunz pasteurianum, and crystallized by Knight and co-workers (6-8). To indicate the functional similarity with ferredoxin, Knight et al. (6) proposed the term “flavodoxin” for their FMN protein. This term has been adopted for similar flavoproteins that were subsequently isolated from a variety of microorganisms (9-18),and it has been extended by some authors 2. L. E. Mortenson, R. C. Valentine, and J. E. Carnahan, BBRC 7,448 (1962). 3. K. Tagawa and D. I. Arnon, Nature (London) 195, 537 (1962). 4. R. M. Smillie, Plant Physiol. 38, 28 (1963). 5. R. M. Smillie, BBRC 20, 621 (1965). 6. E. Knight, Jr., A. J. D’Eustachio, and R. W. F. Hardy, BBA 113, 626 (1966). 7. E. Knight, Jr., and R. W. F. Hardy, JBC 241,2752 (1966). 8. E. Knight, Jr., and R. W. F. Hardy, JBC 242, 1370 (1967). 9. J. LeGall and E. C. Hatchikian, C. R. Acad. Sci., Ser. D 264, 2580 (1967). 10. M. Dubourdieu, J. LeCall, and F. Leterrier, C. R . Acad. Sci., Ser. D 267, 1653 (1968). 11. M. Dubourdieu and J. LeGall, BBRC 38, 965 (1970). 12. S. G. Mayhew and V. Massey, JBC 244, 794 (1969). 13. S. G. Mayhew, BBA 235,276 (1971). 14. M. A. Cusanovich and D. E. Edmondson, BBRC 45, 327 (1971)

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to include not only Smillie’s phytoflavin but also a protein from Azotobacter vinelandii (19,200) , which in earlier publications had been called “free radical flavoprotein” (21,22), “Shethna flavoprotein” (23), or “azotoflavin” (24,25). All of these flavoproteins catalyze the transfer of electrons to and from other proteins, and as they are also broadly similar in their chemical and physical properties, it is appropriate that they should be described by a single term. In this article the general name flavodoxin has been retained because it has been used most often in the literature. As pointed out by Beinert (26) however, this nomenclature is imprecise by comparison with the corresponding term “ferredoxin” for the iron-sulfur proteins. Organisms from which flavoproteins of the flavodoxin type have been isolated include several strictly anerobic bacteria, representatives from the obligately aerobic [ A . vineEandii (19,222)1, facultatively anaerobic [Escherichia coli ( 16 ) ] , and photosynthetic [ Rhodospirillum rubrum (14) ] groups of bacteria, blue-green algae [e.g., Anacystic nidulans (46) and Synechococcus lividus (17)] and a eukaryotic green alga [Chlorella fusca ( 1 6 ) ] (Table IV). They have not yet been found in higher animals and plants. 2. Chemical Composition, Moleculay Weight, and Purification

Flavodoxins contain one equivalent of FMN. Flavin is their only known prosthetic group (5-18) , and they lack transition metals, in particular the iron-sulfur chromophore of the ferredoxins. A tabulation of the amino acid compositions of 12 flavodoxins (27) discloses some general similarities. Acidic amino acids always predominate over basic residues; nine 15. W. G. Zumft and H. Spiller, B B R C 45, 112 (1971). 16. H. Vetter, Jr., and J. Knappe, Hoppe-Seyler’s 2. Physiol. Chem. 352, 433 (1971). 17. H. L. Crespi, U. Smith, L. Gajda, T. Tisue, and R. M. Ameraal, B B A 256, 611 (1972). 18. H. L. Crespi, J. R. Norris, and J. J. Katz, Nature (London), New Biol. 236, 178 (1972). 19. B. van Lin and H. Bothe, Arch. Mikrobiol. 82, 155 (1972). 20. H. Bothe and B. Falkenberg, 2.Naturforsch. B 27, 1090 (1972). 21. Y. I. Shethna, P. W. Wilson, and H. Beinert, B B A 113, 225 (1966). 22. J. W. Hinkson and W. A. Bulen, JBC 242, 3345 (1967). 23. D. E. Edmondson and G. Tollin, Biochemistry 10, 113 (1971). 24. J. R. Benemann, D. C. Yoch, R. C. Valentine, and D. I. Arnon, Proc. Nut. Acud. Sci. U . S.64, 1079 (1969). 25. D. C. Yoch, J. R. Benemann, R. C. Valentine, and D. I. Arnon, Proc. Nut. Acud. Sci. U . S. 84, 1404 (1969). 26. H. Beinert, in “Flavins and Flavoproteins” (H. Kamin, ed.), p. 207. Univ. Park Press, Baltimore, Maryland, 1971. 27. J. L. Fox, S. S. Smith, and J. R . Brown, 2.Naturforsch. B 27, 1096 (1972).

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STEPHEN G. MAYHEW AND MARTHA

L.

LUDWIG

flavodoxins either lack histidine altogether or contain only a single residue. Yet flavodoxins from differentstrains of Desulfovibrio or Clostridium have distinctive compositions (If ,IS). For the flavodoxins surveyed (27), the cysteine content varies from one to five residues and tryptophan from one to six residues. The polypeptide chain lengths, based on amino acid compositions, lie between approximately 120 and 220 residues, corresponding to apoprotein molecular weights between 14,500 and 23,000. According to size, the flavodoxins seem to fall into two groups. One category has molecular weights of 14,500to 17,000, and the other, 20,000 to 23,000. Unless otherwise noted, the molecular weights given in Table IV (Section I1,E) are calculated from compositions. Determinations by ultracentrifugation or gel filtration have occasionally produced somewhat different values. The several published procedures for the purification of flavodoxins exploit their low isoelectric points, which result in retention of these proteins by DEAE-cellulose under conditions where many other proteins are eluted (5,7,12,14,22). Cell-free extracts are often applied directly to DEAE columns. Development with salt gradients results in substantial purification. Ammonium sulfate fractionation and a second DEAE chromatography, followed by gel filtration, provides a highly purified preparation. Several flavodoxins crystallize readily from solutions of ammonium sulfate (7,12,IS,28,29). All are very stable and can be stored for long periods in frozen solution or as crystals a t 4O.

3. Function Flavodoxins do not react directly with small molecules such as the pyridine nucleotides, and their only known biochemical “substrates” are other redox proteins. Nevertheless, the number of reactions known to utilize low potential carriers is impressive; e.g., a recent review (SO) lists 18 ferredoxin-dependent enzymes of fermentative bacteria. Replacement of ferredoxin by flavodoxin has not been attempted in every one of the ferredoxin-requiring reactions, and there are a few systems in which flavodoxins seem unable to fill the role of ferredoxins (3I,S2). However, flavodoxins prove to be efficient carriers in numerous reactions. Smillie (5)was the first to demonstrate that a flavodoxin (phytoflavin from A . nidulam) could replace ferredoxin in the light-dependent reduc28. M. L. Ludwig, R. D. Andersen, S. G. Maphew, and V. Massey, JBC 244, 6047 (1969). 29. K.D. Watenpaugh, L. C. Sieker, L. H. Jensen, J. LeGall, and M. Dubourdieu. ?roc. Nat. Acad. Sci. U . S. 69, 3185 (1972). 30. D. C. Yoch and R. C. Valentine, Annu. Rev. Microbiol. 26, 139 (1972). 31. U. Gehring and D. I. Arnon, JBC 247,6963 (1972). 32. L.L. Barton and H. D. Peck, Bacterial. Proc. p. 134 (1970).

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tion of NADP' by plant chloroplasts. Knight and Hardy (7,8) subsequently showed that flavodoxin from C. pasteurianum is likewise a mediator in the photosynthetic system. I n this chain of reactions, ferredoxin or flavodoxin transfers electrons to the flavoenzyme ferredoxin-NADP+reductase (2,5,33-35). In addition, flavodoxins substitute for bacterial ferredoxins in the phosphoroclastic oxidation of pyruvate (7,8,IZ,gO). Hf

+ CH&OCOO- + HP04'-

+ CH3COOPOs'-

+ COz + Hz

(1) The clastic system in extracts of C. pasteurianum and other anaerobic bacteria includes the enzymes pyruvate dehydrogenase, phosphotransacetylase, and hydrogenase. It utilizes coenzyme A as cofactor; the low potential electron carrier is required to mediate the oxidation of pyruvate dehydrogenase and the reduction of hydrogenase. Reducing equivalents made available by the oxidation of pyruvate can be transferred by ferredoxin or flavodoxin not only to hydrogenase but also to other enzymes that reduce a variety of compounds, including molecular nitrogen (7,19,S6) and pyridine nucleotides (8). Reduced ferredoxin also participates in the reversal of the first step of the clastic reaction, namely, the formation of pyruvate from CO, and acetyl-CoA, catalyzed by pyruvate synthase in various anaerobes and photosynthetic bacteria (S7). Peptostreptococcus elsdenii flavodoxin is able to substitute for ferredoxin in the analogous fixation of CO, into butyrate (38). The dissimilatory pathway of sulfate reduction in Desulfovibrio species relies on ferredoxin or flavodoxin to mediate transfer of electrons between hydrogen and sulfite. Formation of H,S from sulfite proceeds in several steps and the precise role of flavodoxin (or ferredoxin) has not been conclusively established ; the overall reaction is also dependent on cytochrome CRI

(99,401.

I n many cases the requirement for an electron carrier has been established using crude extracts or partially purified enzymes, and for this reason the direct interaction of flavodoxin or ferredoxin with particular A. Son Pietro and H. M. Lang, JBC 231, 211 (1958). M. Shin, K . Tagawa, and D. I. Arnon, Biochem. Z. 338, 84 (1963). M . Shin and D. I. Arnon, JBC 240, 1405 (1965). M. G. Yates, FEBS (Fed. Eur. Biochem. Soc.) Lett. 27, 63 (1972). R . Bachofen, B. B. Buchanan, and D. I. Amon, Proc. Nat. Acad. Sci. U . S . 51, 690 (1964). 38. M. J. Allison and J. L. Peel, BJ 121,431 (1971). 39. E. C. Hatchikian, J. LeGalI, M. Bruechi, and M. Dubourdieu, BBA 258, 701 (1972). 40. K. hie, K . Kobayashi, M . Kobayashi, and M. Ishimoto, J. Biochem. ( T o k y o ) 73, 353 (1973). 33. 34. 35. 36. 37.

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STEPHEN G. MAYHEW AND MARTHA L. LUDWIG

redox enzymes has not always been rigorously demonstrated. An exception is the reaction of carriers with ferredoxin-NADP+-reductase, which has been investigated in considerable detail (Section II,F,4). Purified preparations of nitrogenase have been shown to accept electrons directly from Azotobacter chroococcum flavodoxin (367, but it is not clear whether the site of transfer is the Mo or the nonheme iron moiety of nitrogenase. It has been proposed that a nonheme iron center of the bacterial pyruvate dehydrogenase is oxidized by electron carriers (41) . The relative activities of flavodoxins and ferredoxins as electron carriers have been determined in both plant and microbial systems, with results which indicate that transfer rates vary somewhat according to the source of the carrier; for example, C. pasteurianum and A . niduluns flavodoxins are reported to be twice as active, on a molar basis, as bacterial and A . nidulans ferredoxins, respectively, in stimulating production of NADPPH by washed chloroplasts (5,8). On the other hand, the activity of Chlorella fusca flavodoxin is less than that of Chlorella ferredoxin in the same assay (16). Flavodoxins are generally less efficient than ferredoxins in the phosphoroclastic reaction, although at saturating levels of the carriers the rates become approximately equal (9-17). The interchangeability of carriers with quite different structures and chromophores suggests a lack of recognition in the electron transfer reactions, yet the existence of tight complexes between carriers and ferredoxin-NADP+-reductase has been demonstrated (42-46). Flavodoxin from A . vinelandii differs from other flavodoxins in showing abnormally low activity in several ferredoxin-dependent reactions. As a result some time elapsed before a catalytic function could be ascribed to this protein (21,22). I n 1969, Benemann and co-workers (24) discovered that it is weakly active as an electron carrier between spinach chloroplasts and nitrogenase of A . vinelandii. The activity in nitrogen fixation was confirmed by van Lin and Bothe (19) who showed further that, contrary to earlier indications (14,22,24),this flavodoxin also substitutes for ferredoxin in the photosynthetic reduction of NADP' by plant chloroplasts. The critical difference between the experiments of van Lin and Bothe (19) and previous negative results was the use of an anaerobic gas phase. However, even under these conditions, the catalytic efficiency of this flavodoxin is low, and the maximum rate with saturating concentrations is only about half of the maximum rate observed with fer41. K.Uyeda and J. C. Rahinowitz, JBC 246,3111 (1971). 42. G. P. Foust, S. G. Mayhew, and V. Massey, JBC 244, 964 (1969). 43. N. Nelson and J. Neumann, BBRC 30,142 (1968). 44. M.Shin and A. San Pietro, BBRC 33, 38 (1968). 45. M.Shin, BBA 292, 13 (1973).

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redoxin and flavodoxin from A . nidulans. Bothe and Falkenberg (20) subsequently showed that A . vinelandii flavodoxin can function in the phosphoroclastic oxidation of pyruvate catalyzed by extracts of C . pasteurianum. The principal metabolic role of A . vinelandii flavodoxin still remains to be established. Enzyme assays based on the activity of flavodoxin in the photosynthetic reduction of NADP’ (46), the production of hydrogen from dithionite in the presence of hydrogenase ( 7 ), and the phosphoroclastic oxidation of pyruvate (12) have been described. These are unsatisfactory in several respects: first, they are insensitive; second, they depend on crude and unstable preparations of other fractions; third, they do not distinguish between flavodoxin and ferredoxin, and consequently flavodoxin can be positively identified only after it has been obtained in pure form. More recently, in the authors’ laboratories, flavodoxin has been assayed by its ability to couple NADPH oxidation to cytochrome c reduction in the presence of purified ferredoxin-NADP+-reductase ( 4 7 ) . This procedure still suffers from the lack of discrimination between flavodoxin and ferredoxin. Enzymic assay is of only limited use during routine purification, and purity can usually be more reliably estimated from the absorption spectrum (Section II,D,l) . However, estimates of the quantities of flavodoxin and ferredoxin in crude extracts have necessitated their preliminary separation, and are therefore uncertain, particularly in the case of the more unstable ferredoxins. The lack of a suitable catalytic assay might be circumvented by the use of immunochemical techniques. It has been found that antibodies to flavodoxins from P. elsdenii and Clostridium M P do not cross react with ferredoxins from these sources (48)*

4. Regulation by Iron I n certain microorganisms the synthesis of flavodoxin occurs only during growth in iron-poor media. This regulation by iron is the first of its kind observed with a flavoprotein, though similar effects on the synthesis The effect of iron of flavin have been known for about 30 years (49,50). on C. pasteurianum was noted by Knight and Hardy ( 7 ) , who showed that little if any ferredoxin is produced by this organism under iron-deficient conditions. Knight and Hardy ( 7 ) concluded that the flavoprotein 46. R. M. Smillie and B. Entsch, “Methods in Enzymology,” Vol. 23, Part A, p. 504, 1971. 47. M. Shin, “Methods in Enzymology,” Vol. 23, Part A, p. 440, 1971. 48. H. J. Somerville and S. G. Mayhew, unpublished. 49. R. J. Hickey, Arch. Biochem. 8, 439 (1945). 50. A. L. Demain, Annu. R e v . Microbial. 26, 369 (1972).

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STEPHEN G. MAYHEW AND MARTHA L. LUDWIG

is synthesized as a replacement for ferredoxin when the metal is in short supply. Iron has a similarly pronounced effect on the synthesis of ferredoxin and flavodoxin in P , elsdenii. The use of immunochemical techniques to estimate flavodoxin in cell-free extracts of P. elsdenii has permitted the conclusion that iron-rich cells do not contain flavodoxin or its apoprotein, and that iron deficiency brings about de nouo synthesis ( 4 8 ) .I n other organisms control by iron is less dramatic, and it is more difficult to obtain cells either free of ferredoxin in iron-poor media (51) or of flavodoxin in iron-rich media (39,40). In still a third group of organisms, E. coli and A . uinelandii, flavodoxin synthesis appears to be independent of iron (16‘,19,24). It is not clear, however, whether the ratio of ferredoxin to flavodoxin in these two organisms is sensitive to iron, nor is it known whether flavodoxin and ferredoxins from A . vinelandii, and other organisms in which proteins of both types are synthesized simultaneously, share the same functions. The picture is further complicated in the case of A . uinelandii by the presence of more than one type of ferredoxin (19,25,62). The iron concentration which favors flavodoxin synthesis varies with the organism, but is in the range of 0.01-0.5 pg/ml. The iron restriction markedly limits the total growth of certain organisms [ e.g., Clostridium MP (13)] and in such cases it is clear that flavodoxin synthesis depends on a true iron deficiency. The total growth of other organisms is much less affected (e.g., 7,12).However, batch cultures have been used in investigations on the relationship between iron levels and flavodoxin synthesis, and it is not known whether the protein is synthesized throughout growth or only when the available iron is depleted. There is surprisingly little information about the overall effects of iron deficiency on organisms which require low concentrations of iron for the production of flavodoxin. Clostridium pasteurianum is less rodlike, more ovoid, and almost white in iron-deficient media, and anaerobic cells of P. elsdenii from iron-poor media are gray in contrast to cells from ironsufficient media, which, depending on the excess of iron, are green-brown or black ( 5 3 ) . Cells of C . pasteurianum which contain flavodoxin still catalyze the reduction of molecular nitrogen (79, and in P. elsdenii the overall fermentation of lactate to fatty acids, hydrogen, and carbon dioxide (54) is not appreciably influenced by iron deprivation ( 5 3 ) .It is pos51. H. Bothe, P. Hemmerich, and H. Sund, in “Flavins and Flavoproteins” (H. Kamin, ed.), p. 211. Univ. Park Press, Baltimore, Maryland, 1971. 52. Y. I. Shethna, D. V. Dervartanian, and H. Beinert, BBRC 31, 862 (1968). 53. S. G. Mayhew, unpublished. 54. S. R. Elsden, B. E. Velcani, F. M. C. Gilchrist, and D. Lewis, J. Bacterial 72, 681 (1956).

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65

sible therefore that iron proteins such as nitrogenase (55), hydrogenase (56),and pyruvate dehydrogenase (41) are not drastically affected under these conditions, but this point has not been tested with the purified proteins. However, there is evidence that some iron proteins are more critical than others ; for example, several iron-deficient organisms contain the iron protein rubredoxin (12,lS), and Chlorella fusca from iron-poor medium still contains cytochromes and nitrite reductase (15). I n view of the profound effects of iron on flavin synthesis by some microorganisms (49,50),it is interesting that the total flavin content of P. elsdenii (57) is not appreciably affected by mild iron deficiency; F M N is increased, but FAD is correspondingly lower (53). 5. Some Properties of the Bound Flavin Mononucleotide In all the flavodoxins the oxidation-reduction potentials of bound F M N differ significantly from those for the free prosthetic group. The two oneelectron steps have distinct potentials with the consequence that the semiquinone form can be obtained in essentially quantitative yields under appropriate conditions. Redox potentials for the semiquinone-fully reduced couple are in the range typical of ferredoxins (Table IV, Section II,E), and are the lowest known for any flavoprotein. Potentials for the oxidized-semiquinone couple are frequently higher than for free FMN. From these data it seemed reasonable to postulate that in vivo the flavodoxins may act as one-electron carriers, shuttling between the fully reduced and semiquinone states (19,58), and there is some evidence to support this suggestion ( 3 6 ) . The prosthetic group is bound tightly but not covalently by apoflavodoxins. The measured association constants are of the order of lo8 or greater, but the holoproteins are reversibly dissociated by a number of procedures used to prepare other apoflavoproteins, such as low p H or dialysis against concentrated KBr (5,59-61). A limited number of modified flavins can be bound instead of FMN, with the specificity depending upon the species from which the flavodoxin is derived (59,61) (Section II,C,3). Spectroscopic differences among the flavodoxins suggest that the environment of the flavin chromophore varies with the species. It has been proposed that the flavodoxins be classified into two spectral groups, one that resembles C. pasteurianum flavodoxin, and a second group, in55. H. Dalton and L. E. Morteneon, Bacterial. Rev. 36, 231 (1972). 56. G. Nakos and L. E. Mortenson, Biochemistry 10,2442 (1971). 57. J. L. Peel, BJ 69, 403 (1958). 58. S. G. Mayhew, G. P. Foust, and V. Massey, JBC 244,803 (1969). 59. S. G. Mayhew, BBA 235, 289 (1971). 60. J. W. Hinkson, Biochemistry 7, 2666 (1968). 61. D. E. Edrnondson and G. Tollin, Biochemistry 10, 124 (1971).

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STEPHEN G. MAYHEW AND MARTHA L. LUDWIG

cluding D. vulgaris flavodoxin, that more closely resembles flavodoxin from R. rubrum (62). This division does not coincide with that based on molecular weight. In many respects the flavodoxins resemble the larger flavoprotein dehydrogenases (63).Both classes of proteins form a blue neutral semiquinone rather than the red anion; they resist substitution by sulfite a t N-5; and they are reoxidized by oxygen in two steps with concomitant formation of the superoxide radical.

B . STRUCTURES 1. Introduction At the present time the detailed three-dimensional structures of two flavodoxins, those from D. vulgaris (29,64) and Clostridium MP (65,66), are known, along with the corresponding amino acid sequences (67,68). For Clostridium M P flavodoxin, electron density maps of not only the oxidized (66) but also the semiquinone and reduced states (65,69) have been computed at high resolution. In addition, the complete sequence of P. elsdenii flavodoxin (70) and partial sequences of Clostridium pasteurianum (27,7l) flavodoxin have been reported. From the sequences alone, it appears that one region near the N-terminus (part of the FMN binding site) is highly conserved during evolution, but farther along the chain homologies are more difficult to discern by inspection. Nevertheless, the three-dimensional folding of D. vulgaris and Clostridium hilP flavodoxins clearly demonstrates structural homology. The surprising conclusion from 62. J. A. D’Anna, Jr. and G. Tollin, Biochemistry 11, 1073 (1972). 63. 8. Massey, F. Muller, R. Feldberg, M. Schuman, P. A. Sullivan, L. G. Howell, S. G. Mayhew, R. G. Matthews, and G. P. Foust, JBC 244, 3999 (1969). 64. K. D. Watenpaugh, L. C. Sieker, and L. H. Jensen, Proc. Nut. Acad. Sci. U. S. 70, 3857 (1973). 65. R. D.Andersen, P. A. Apgar, R. M. Burnett, G. D. Darling, M. E. LeQuesne, S. G. Mayhew, and M. L. Ludwig, Proc. Nut. Acad. Sci. U . S. 89, 3189 (1972). 66. R. M. Burnett, G. D. Darling, D. S. Kendall, M. E. LeQuesne, S. G. Mayhew, W. W. Smith, and M. L. Ludwig, JBC 249,4383 (1974). 67. M. Dubourdieu, J. LeGall, and J. L. Fox, BBRC 52, 1418 (1973). 68. M. Tanaka, M. Haniu, K. T. Yasunobu, and S. G. Mayhew, JBC 249, 4393 (1974). 69. M. L. Ludwig, R. M. Burnett, G. D. Darling, S. R. Jordan, D. S. Xendall, and W. W. Smith, in “Structure and Conformation of Nucleic Acids and ProteinNucleic Acid Interactions” (M. Sundaralingam and s. T. Rao, eds.) (in press). 70. M. Tanaka, M. Haniu, K. T. Yasunobu, S. G. Mayhew, and V. Massey, JBC 248, 4354 (1973); 249, 4397 (1974). 71. M. Tanaka, M. Haniu, G. Matsueda, K. T. Yasunobu, S. G. Mayhew, and V. Massey, Biochemistry 10, 3041 (1971).

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comparisons of the two structures is that many of the variations between species occur a t the active center, in the vicinity of the isoalloxazine ring. Structural differences in the neighborhood of the flavin ring are compatible with the distinctive spectral properties of the two proteins (Section II,D,l). 2. Determination and Comparison of Chemical Sequences

Total sequence determination has so far been confined t o the smaller flavodoxins with chain lengths less than 150 residues. These proteins have presented no extraordinary problems, yielding tractable peptides after cleavage with CNBr, trypsin, chymotrypsin, and thermolysin (67,68,7O). Sequences as long as 52 residues have been determined by automated Edman degradation (68). The primary structures of Clostridium M P and D. vulgaris flavodoxins, comprising 138 and 148 residues, respectively, are displayed in Fig. 1. Some of the residues determined to be equivalent (Le., structurally homologous) by comparison of the three-dimensional models are also shown. The chain folding in the two flavodoxins (Section II,B,Q) has been compared after applying to one model the rigid-body rotations and translations required to minimize the squares of the distances between related &-carbon atoms (72). (Nonequivalent atoms are eliminated as the calculation proceeds.) Preliminary calculations have established the general similarity of the helical and sheet domains in the two structures ( 6 5 ) . Identification of all residues which occupy equivalent positions in the three-dimensional structures is incomplete a t the time of writing, but homologies based on the correspondence of C, positions have been established for the p sheet and the N-terminal helix (Figs. 1-3). The results indicate the regions in which the extra 10 residues are inserted in the D. vulgaris chain. Three additions occur a t the N-terminus, one more somewhere between residues 38 and 47, two between 57 and 77, three between 89 and 100, and one beyond 123. (Numbers refer to the Clostridium MP sequence.) Superposition of the drawings in Fig. 2 reveals several areas in which the matching of the structures is imperfect, notably in the vicinity of Clostridium MP residues 40, 58, and 90, and along much of the helix surrounding residue 70. The primary sequences of the flavodoxins have been analyzed to determine those residues which are homologous in the evolutionary sense (67,73). Since the C-terminal portions of the flavodoxin chains, from approximately residue 90 (Clostridium M P numbering) onward, are highly variable, alignments have relied in part on the principle of mini72. s. T. Rao and M. G. Rossmann, J M B 76, 241 (1973). 73. W. M. Fitch and K. T. Yasunobu, private communication.

68

STEPHEN G. MAYHEW AND MARTHA L. LUDWIG 1 10 Met Lys Ile Val T y r T r p Ser Gly T h r Gly Asn T h r Glu Lys Met Ala Glu Leu Ile Ala 21 30 40 Lye Gly Ile Ile Glu Ser Gly LYEAsp Val Asn T h r Ile Asn Val Ser Asp Val A m Ile 41 50 60 Asp Glu Leu Leu Asn Glu Asp Ile Leu Ile Leu Gly CyS S e r Ala Met Gly Asp Glu Val 80

70

61

Leu Glu Glu Ser Glu Phe Glu P r o Phe Ile Glu Glu Ile S e r T h r Lys Ile Ser Gly Lys 81 go 100 Lys Val Ala Leu Phe Gly Ser Tyr Gly T r p Gly Asp Gly Lys T r p Met Arg Asp Phe Glu 101 110 Glu Arg Met Asx Gly T y r Gly CyS Val Val Val Glu T h r P r o Leu Ile Val Gln

120

+ Glu

121 130 138 P r o Asp Glu A h Glu Gln Asp CyS Ile Glu Phe Gly Lys Lys Ile Ala Am Ile

(a)

CLDST.RlDlUM

Up 1 I I

0. VULGARIS C. PASTEURIANUM P. ELSDENll

(21

D. VULGARIS

MP 1 I 1 ( 21

CLOST~IOIUM MP II

I

(21

C. PASTEURIANUM P. ELSOENII CLOST.RlDlUM

D. VULGARIS

30

-8-

M*+VEIVYWSGTGNTEAMANEIEAAVKAAGADVESVRF

C. PASTEURIANUM P. ELSDENII

D. VULGARIS

-

20

40

M*KVNIIYWSGTGNTEAMAKLIAEGAQEKGAOVKLLNV x)

CLDST!tlPlUM

-

I0

-8-

M*K**IVYWSGTGNTEKMAELIAUGI I E S G K D V N T I N V S D I371 MKIIVYWSGTGNTEKMAELIAKGIIESGKDVIMTINVSD M P K A L I V Y G S T T G N T E Y T A E T I A R E L A ~ A G Y E V D S R D A A1401 S

YP 1 II ( 21

m

60

-8-

-a-

ED

VNIDELLNE*DILILGCSAMGDEVLEESEFfPFlEElSTK (761 V DI L I L G C 5 AMGI V E A G G L F E G F D L V L L G C S T W G D D ~ OI * * L Z B B F I P L F D S L 1781 DVVAFGSPSMG DVILLGCPAMG 90 100 I10 I20

**

-B-

-

-a-

I S G K K V A L F G S Y G W G O* ISGKKVALFGSYG

* G K W M R D F E E R M N G Y G C V il091 E E R M N G Y GCV

Z Z T G A Z G R K V A C F G C G B S S Y E Y F C G A V D A I E E K L K N L G A Z 11181 GKKEGAFXXXX GKKVGLFGSYG

8

I30

-

-a-

I40

ISD

VVETPL*IVONEPOEAEQD.CI EFGKKlANl V V E T P L I V O W E P DIE1 I VLBGLR I DGDPRAARBB IVGWAHDVRGA I

(1381 (1481

(b)

FIQ.1. (a) The sequence of Clostridium M P flavodoxin. Sequences forming the FMK binding site are underlined; that does not imply that every underlined residue provides contacts with the prosthetic group (cf. Figs. 4 and 5 ) . (b) Comparison of the chemical sequences of four flavodoxins, employing a numbering scheme which allowa for deletions (*) in each chain. The actual residue number a t the end of each line is given in parentheses. The sequence of C. pasteuriunum flavodoxin is unknown between position 93 and the seven residues at the C-terminus. Lines (1) and (3) through ( 5 ) display the alignments which minimize the number of mutations required to relate the four sequences (75). Lines (2) and (3) show some of the structurally equivalent a-carbons, determined by rigid-body fitting of the coordinates. When the structures are superposed, each C, in line (2) is less than 2.0 A from the corresponding atom in the D. vulgaris structure, except for those few residues enclosed in parentheses. These latter C , atoms are in matching regions and are separated by just slightly more than 2.0 A. The average distance between the equivale'nt atoms shown is 1.1 A. A number of additional equivalent a-carbons have been omitted from line (2), pending comparison of the p-carbon positions. The location of the p structure and of helices in Clostridium M P flavodoxin is indicated by arrows. Residues constituting the F M N binding site of D. vulgaris flavodoxin are shown in Fig. 4b.

2.

FLAVODOXINS AND ELECTRON-TRANSFERRING

FLAVOPROTEINS

69

mum mutation frequency ( 7 4 ) . At many positions, including much of the p sheet, the alignments determined from the structures are consistent with those expected from mutation of a “primordial” flavodoxin gene. The approximate location of gaps and insertions is in agreement. However, the homologies deduced by the two methods do not always coincide. The N-terminus provides a nice illustration of the discrepancies. Althmgh the two methionines presumably initiate both chains and are related according to genetic analysis, i t happens that deletions in the Clostridium MP chain can be accommodated in the structure by placing Met-1 a t the position occupied by the fourth residue of the D. vulgaris chain. Again, in the final strand of p sheet, genetic arguments suggest an insertion in the D. vulgaris chain a t position 127 of Fig. lb, following Leu-115 of Clostridium M P flavodoxin. There is no corresponding deviation of the superposed structures at this point in the p sheet; instead the chains remain in register for this and several succeeding residues. Correspondence of the two structures is poor for at least seven residues of the D. vulgaris chain, beginning with Ser-96, even though this sequence constitutes part of the active site and the residues may be derived from the same ancestor. The disparity between structural equivalences and the alignments predicted from genetic considerations recalls similar difficulties in assignment of homologies in cytochrome c ( 7 5 ) . Comparison of the sequences of C . pasteurianum and P. elsdenii flavodoxins with those of Clostridium M P and D. vulgaris flavodoxins demonstrates that the first three proteins are more closely related t o one another than to D. vulgaris flavodoxin (67,7’3).Only portions of the known sequences of C . pasteurianum and P. elsdenii flavodoxins have been selected for Fig. l b ; these correspond to the most highly conserved parts of the structure, where the relationships of the sequences are often evident by inspection. At present there is no information on the location of the extra residues in the flavodoxins of molecular weight near 20,000. It will be fascinating to see whether these longer chains contain additional elements of secondary structure (cf. Volume XI, Chapter 2). Evolutionary conservation of most of the residues a t positions 6 through 16 is obvious from Fig. lb. Several other positions, ie., Ala-19, Ile-22, Gly-30, Val-33, Asp-51, Gly-56, Gly-61, Gly-87, Lys-89, and Phe-93, are invariant in the portions of the four different flavodoxins shown in Fig. lb. The region from position 10 to 15, containing several hydroxyamino acids, constitutes part of the binding site for the phosphate portion of the flavin mononucleotide. However, many of the other active center residues of Clostridium M P and D . vulgaris flavodoxin differ. The 74. W. M. Fitch, J M B 49, 1 (1970). 75. R.E. Dickerson, J M B 57, 1 (1971).

70

STEPHEN G. MAYHEW AND MARTHA L. LUDWIG

relative arrangements of polypeptide and FMN in each molecule are described more explicitly in the following discussion of the three-dimensional structures. Although modification of cysteine residues is known to interfere with FMN binding in the four flavodoxins included in Fig. l b (8,13,40,69), each cysteine is replaceable. 3. Three-Dimensional Structures

a. Structure Determination. The pertinent crystallographic data for Clostridium M P and D. vulgaris flavodoxins are given in Table I. Despite their conformational similarities, the molecules of Clostridium M P and D. vulgaris flavodoxins pack quite differently in their respective unit cells, with the crystals of the D . vulgaris species containing relatively more solvent. Phases for Clostridium MP flavodoxin were calculated using SrnIII (76) and Au1 (66,66)derivatives and incorporating anomalous scattering from each, but Watenpaugh et al. (29,64) have demonstrated that the Sm"' derivative alone, thanks to the large anomalous scattering TABLE I CRYSTALLOQRAPHIC DATAFOR FLAVODOXINS Space group

Flavodoxin

D. vulgaris oxidized

Clostridium MP oxidizedd

Cell dimensions

P43212 a = b = 51.6 c = 139.6 P3121 a = b = 61.56 c = 70.36

Clostridium MP,

P3121

a

Clostridium MP,

P3121

a

semiquinone reduced!

c c

~~

Resolution

(b)

(m).

Heavy atoms

2.0

0.74-0.68* Sm"1 at Glu-25, ASP-63" 1.9' 0.81 Sm"1 at Glu-123, ASP-124 AuI a t Cys-53, CYS-128 0.72 = b = 61.63 2.08 = 70.98 = b = 61.68 2.5 = 71.05

~~~~

Mean figure of merit. The variation from the innermost to outermost resolution ranges. From Jensen and Watenpaugh (78). Partial refinement of this structure has reduced the crystallographic R factor to 0.27. The smallest intensities, representing about 15% of the scattering, have not been included in the maps. Difference map, with coefficients m(lFlr.d - IFI.,) exp ia., (79).

76. M. L. Ludwig, R. D. Andersen, P. A. Apgar, R. M. Burnett, M. E. LeQuesne, and S. G. Mayhew, Cold Spring Harbor Symp. Quant. Biol. 36, 369 (1972).

2.

FLAVODOXINS AND ELECTRON-TRANSFERRING FLAVOPROTEINS

71

by this element, can suffice for phase determination. Sm"' has provided a suitable heavy atom derivative for still another highly acidic protein, bacterial ferredoxin (7'7), and is observed in all three structures to bind at Asx and Glx residues. However, the Sm positions are not identical in the two flavodoxin structures (Table I). One-electron reduction of both crystalline flavodoxins is accompanied by rather large changes in the diffracted intensities (28,'78). R I = 2110x- I s q l / 2 ( I o x I s , ) is 0.33 for the Clostridium M P data to 2 A resolution. Because of the magnitude of the intensity differences, the phases for the semiquinone form of Clostridium M P flavodoxin have been determined independently, utilizing the same heavy atom substituents (65,699).The average difference in phase angle, determined by isomorphous replacement, is 5 9 O . (Uncorrelated phases would give a value of goo.) Examination of the structures shows that for Clostridium M P flavodoxin the intensity differences arise from a combination of conformational changes with an overall (rigid-body) motion of the molecules in the unit cell. The X-ray and chemical sequence studies of Clostridium M P flavodoxin were complementary. In the map of the oxidized form obtained by isomorphous replacement phasing of 1.9 A data, more than 20 residues were insufficiently defined to permit their identification. Six of these residues occur in the region 36-48, adjacent to a large channel of solvent, and five others at 109-114 and 122-124, also at the surface of the molecule. Identification of residues 88-96 in the map clarified the sequence of this segment and observation of the branch points on the side chains discriminated between Leu and Ile a t residues 49 and 50. At the present time there are no known discrepancies between the chemical sequence and the electron density map. b. Molecular Conformation. The drawings of Fig. 2 depict the folding of the polypeptide chain and the relative orientation of the prosthetic group in oxidized Clostridium MP and D. vulgaris flavodoxins. The close similarity of the two structures is evident. Both flavodoxins are characterized by a high proportion of secondary structure; in each molecule a central parallel p sheet is flanked on either side by pairs of helices. No antiparallel sheet is found, but the chain changes direction a t a num-

+

77. E. T. Adman, L. C. Sieker, and L. H. Jensen, JBC 248, 3987 (1973). 78. L. H. Jeneen and K. D. Watenpaugh, private communication. 79. IFI.., IFlaq, I F I I C d , structure factor amplitudes for oxidized, mmiquinone, and reduced crystals, respectively, referred to as IFIObs when the oxidation state is obvious; a,, or as,,, phases for the oxidized or semiquinone structures, determined by isomorphous replacement; IFIc.lc and a,.le, structure factor amplitude and phase, respectively, computed from the atomic positions; m, figure of merit.

72

STEPHEN G. MAYHEW AND MARTHA L. LUDWIG

FIQ.2. Drawings of the C, and FMN atoms of (a) oxidized Clostridium MP flavodoxin and (b) oxidized D . vuZguriS flavodoxin. In (a), C. of residue 115 eclipses C, 114. Residue 1 of the D. vulgaris chain is omitted since its image does not appear in the electron density (78). The models are shown in approximately the same orientation with respect to the protein atoms, and hence the different arrangement of the isoalloxaaine ring is emphasized. (a) is from Burnett et ul. (66).

ber of hydrogen-bonded 3,0 bends (80). The hydrogen bonding schemes for the p sheet, shown in Fig. 3, are based on coordinates obtained from the isomophous replacement maps. I n Clostridium MP flavodoxin, the following residues appear to contribute a t least one hydrogen bond for helix formation: 10-27, 66-74, 93-107, and 124-138. Deviations from the helix have been noted for certain of these residues (66). According to the skeletal model constructed from the 1.9 A map, the number of residues assigned to helices, 310 bends, or p structure is 115 of the total 138 in Clostridium MP flavodoxin (66). Examination of the p sheets and flavin binding regions of the two molecules reveals the importance of water in maintenance of the structures. In two locations the regular sheet hydrogen bonding is interrupted by bonding to solvent or side chains (Fig. 3). The D.vulgaris molecule sub80. C . M. Venkatachalam, Biopolymers 6, 1425 (1968).

2. FLAVODOXINS

AND ELECTRON-TRANSFERRING FLAVOPROTEINS

73

FIG.3. Hydrogen bonding schemes proposed for the parallel /3 sheet found n Clostridium MP and D. vulgaris flavodoxins (66,SS). The residues of Clostridium M P flavodoxin are shown above, in larger type; the equivalent D. vulgaris residues are beneath in smaller lettering. Water molecules and hydrogen bonds found only in D. vulgaris flavodoxin are represent.ed by smaller letters and dotted bonds, respectively. Dashed hydrogen bonds are common to both structures with the exception of the bond from Asx-122 to water, which does not occur in D . vulgaris flavodoxin.

stitutes a solvent interaction for the hydrogen bond to Trp-95 which occurs in Clostridium M P flavodoxin. Similar exchanges of solvent for side chain interactions can be observed in Fig. 4. A water near 0-1 in Clostridium M P flavodoxin is "replaced" by Trp-60 in the D. vulgaris molecule; similarly, a solvent molecule bridging the ribityl 0-4' and the carbonyl o f residue 128 in D. vulgaris flavodoxin is the counterpart of the side chain of Ser-87 in the Clostridium M P structure. Comparisons of the folding of D. vulgaris and Clostridium M P flavodoxins with several pyridine nucleotide dehydrogenases have suggested that the flavodoxins may be members of a larger family of nucleotidebinding proteins (81,82). Appropriate superposition of the parallel sheets of flavodoxin and lactate dehydrogenase brings the F M N phosphate into approximate correspondence with the adenine phosphate of NAD' and aligns the first, second, and third helices of flavodoxin with helices a B , CrE, and cwF of LDH, respectively. However, the positions of the flavin and nicotinamide rings do not quite coincide in this superposition. The 81. M. G. Rossmann and A. Liljas, J M B 85,177 (1974). 82. M. G. Rossmann, D. Moras, and K. W. Olsen, Nature (London) 250, 194 (1974).

74

STEPHEN G. MAYHEW AND MARTHA L. LUDWIG

A

3

(b) FIG.4. Stereo view of the FMN-binding sites of (a) oxidized Clostridium M P and (b) oxidized D. vulgaris flavodoxins. The two drawings have been oriented to provide approximately the same view of equivalent protein atoms. Some bound solvent atoms appear in both drawings. The hydrogen bonding scheme for D. vulgaris flavodoxin, included in (b) can be compared with that for Clostridium M P flavodoxin, shown in Fig. 5.

homologies between the flavodoxins and dehydrogenases are presented and discussed in more detail in Chapter 2, Volume XI. c. T h e Flavin Mononucleotide Binding Site. Stereo views of the two FM N binding sites are presented in Fig. 4, and the probable hydrogen bonding interactions between F M N and protein in Figs. 4 and 5 and Table I1 (69, 83-86). In both structures the isoalloxazine ring is found 83. K. D. Watenpaugh, L. C. Sieker, J. R. Herriott, and L. H. Jensen, Acta Crystallogr., Sect. B 29, 943 (1973). 84. R. Diamond, Acta Crystallogr. 21, 253 (1966). 85. R. Diamond, Acta Crystallogr., Sect. A 27, 436 (1971).

2.

FLAVODOXINS AND ELECTRON-TRANSFERRING FLAVOPROTEINS

75

FIG.5 . Proposed hydrogen bonding contiibutions to the FMN-protein interactions in Clostridium MP flavodoxin. The orientation is shifted slightly from Fig. 4a. For the flavin ring and the ribityl side chain, the bonds indicated by (-.-) are those which seemed most likely according to the initial model, before refinement. The bonds to phosphate oxygens were selected to illustrate the similarity t o the interactions in D. vulgaris flavodoxin. As can be seen from Table 11, alternative or additional hydrogen bonds are possible.

at the periphery of the molecule with the dimethylbenzene end accessible to solvent and the pyrimidine portion “buried” in the protein. Two segments of the polypeptide chain, residues 56-59 and 89-91 in Clostridiurn M P or 60-62 and 95-102 in D. vulgaris flavodoxin provide interactions with the isoalloxazine ring. The ribityl side chain extends toward the interior of the protein (P:N-10 = 8.5 A in Clostridium M P flavodoxin) , permitting OH-2’ and OH-4’ to form hydrogen bonds with protein atoms. The predominant contribution to the binding of the phosphate moiety is made by residues 7-12 (1C15 in D. vulgaris flavodoxin), which include the initial turn of an a-helix. In both structures the resolution is sufficient to determine the P-0 directions. The conformation of the protein-bound FMN, in terms of the torsion angles along the ribityl-phosphate side chain, is similar to that observed in model structures (69,86). A difference in the torsion angles at N-10 to C-1’ correlates with the dissimilar orientations of the isoalloxazine rings in D. vulgaris and Clostridiurn M P flavodoxins (see below) ; otherwise, the FMN conformations in the two structures are very similar. From C-1’ to C-4’ the chain is in a trans, extended conformation, but close contact of 0-2’ and 0-4’ is avoided by a rotation of the (2-3’ to C-4’ bond (x = 60° rather than B O O ) . The dihedral angles for C-4’ to 86. K. D. Watenpaugh and L. H. Jensen, in “Structure and Conformation of Nucleic Acids and Protein-Nucleic Acid Interactions” (M. Sundralingam and S. T. Rao, eds.) (in press).

76

STEPHEN G . MAYHEIW AND MARTHA L. LUDWIG

DISTANCES BETWEEN FMN

AND

TABLE I1 PROTEIN ATOMS”IN Clostridium M P FLAVODOXIN ~

FMN atom

0-1

0-11

0-111

0-4’

~~

~~

~

~~~

Distance

FMN atom

Protein or solvent atom

0-3’

W-2 w-3

2.7 3.0

w-lb

2.5 2.8 2.7

Ser-7 OH Thr-12 NH Asn-11 NH Thr-12 OH Gly-8 NH

2.7 2.8 3.6 3.3 3.7

0-2’

Ala-55 CO

2.9

N-1

Gly-89 NH

2.9

0-2

Thr-9 OH Thr-9 NH Gly-10 NH Asn-11 NH Ser-7 OH Asn-11 (NH1,O) Gly-8 NH

2.9 3.0 3.4 3.2 3.4 3.7 3.7

Gly-91 NH Gly-89 NH Trp-90 NH w-4

2.8 2.9 3.4 3.3

N-3

Glu-59 COO-

2.8

0-4

Glu-59 NH Asp-58 NH

2.9 3.4

Ser-87 OH Asn-11 (NH2,O) Asn-119 (NH,,O)

2.6 3.0 3.6

N-5

Asp-58 NH

4.2

Protein or solvent atom Gly-8 NH Ser-54 OH

(A)

Distance

(A)

Selected heteroatoms in the vicinity of flavin atoms. Most of the listed atoms are displayed in Fig. 5, but every pair does not necessarily form a hydrogen bond. Distances were calculated from protein coordinates obtained after four cycles of difference FMN coordinates Fourier refinement (83),followed by real space refinement (69,86). were determined by real space refinement (89,86). Although designated &B water, this peak could represent an NHI+ ion.

C-5’ and C-5’ to 0-5’ produce approximately trans conformations about these bonds, and the phosphate oxygens are partly staggered with respect to the C-5’ to 0-5’ bond. As in certain riboflavin structures (87,88), the ribityl 0-2’ is approximately cis to the flavin N-1. The isoalloxazine ring appears to be planar, as expected for the oxidized state (89). When the 87. T. D. Wade and C. J. Fritchie, Jr., JBC 248, 2337 (1973); W. T. Garland, Jr., and C. J. Fritchie, Jr., JBC 249, 2228 (1974). 88. D. Voet and A. Rich, in “Flavins and Flavoproteins” (H. Kamin, ed.), p. 23. Univ. Park Press, Baltimore, Maryland, 1971. 89. P. Kierkegaard, R. Norrestam, P.-E. Werner, I. Csoregh, M. von Glehn, R. Karlsson, M. Leijonmarck, 0. Ronnquist, B. Stensland, 0. Tillberg, and L. Torbjornsaon, in “Flavins and Flavoproteins” (H. Kamin, ed.), p. 1. Univ. Park Press, Baltimore, Maryland, 1971.

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77

extent of folding about the N-5:N-lO direction was estimated from the electron density of oxidized Clostridium M P flavodoxin, using real space refinement ( 8 5 ) ,the angle between the dimethylbenzene and pyrimidine planes was found to be less than 2 O (69). The orientation of the protein about the phosphate group appears identical in the two flavodoxins. Four homologous hydroxyamino acids and five backbone NH groups are near the phosphate oxygens. The environment of the phosphate is remarkable in several respects. First, the phosphate is partially buried in a region devoid of countercharges. I n neither structure are there any neighboring basic residues to compensate for the charge on the phosphate, presumably bound as either the mono- or dianion (90). The environment is quite unlike that observed for NAD’ in lactate dehydrogenase (91), or for nucleotides bound to ribonuclease A (9.2)or to staphylococcal nuclease (9S),where arginine or lysine residues are adjacent to the phosphate. The solvent or counterion access to phosphate oxygens has not been systematically computed (94), but appears to be limited. I n Clostridium MP flavodoxin, only one of the three oxygens seems able to form hydrogen bonds to solvent without displacing protein atoms. A solvent molecule is observed about 2.7 A from this oxygen (Table 11).In the D.vulgaris structure this position is occupied by atoms of Trp-60. Second, the phosphate binding site is conserved, despite the differences in the isoalloxazine interactions in the two structures. Finally, the phosphate group appears to be essential for association of flavins with Clostridium M P and P. elsdenii flavodoxins, whereas D.vulgaris flavodoxin readily binds riboflavin (Section II,C13). The ribityl side chain interactions are similar though not identical in the two structures. A backbone carbonyl to OH-2’ hydrogen bond occurs in each molecule, as does an interaction between 0-3’ and solvent and between Asn-11 (or Asn-14) and 0-4’. The other 0-4’ interactions differ slightly (Figs. 4 and 5). Contrasts in the isoalloxazine-protein interactions are striking. I n Fig. 4 the drawings are oriented to optimize matching of the protein atoms; when the protein “overlaps” are maximized, the flavin rings are found to be inclined to one another at an angle of - 2 4 O (96). Both flavin rings 90. M. L. MacKnight, J. M. Gillard, and G . Tollin, Biochemistry 12, 4200 (1973). 91. M. J. Adams, M. Buehner, K. Chandrasekhar, G. L. Ford, M. L. Hackert, A. Liljas, M. G. Rossmann, I. E. Smiley, W. S. Allison, J. Everse, N. 0. Kaplan, and S.G. Taylor, Proc. Nat. Acad. Sci. U . S. 70, 1968 (1973). 92. F. M. Richards and H. W. Wyckoff, “The Enzymes,” 3rd ed., Vol. 4, p. 647, 1971. 93. F. A. Cotton and E. Hazen, Jr., “The Enzymes,” 3rd ed., Vol. 4, p. 153, 1971. 94. B. Lee and F. M. Richards, J M B 55, 379 (1971). 95. The estimated error in positioning each ring is 2 3 ” .

78

STEPHEN G . MAYHEW AND MARTHA L. LUDWIG

are sandwiched between hydrophobic residues, but these residues differ in the two flavodoxins (Figs. 4 and 5). In Clostridium M P flavodoxin, they are Met-56, toward the interior of the molecule, and Trp-90, which partially shields the flavin ring from solvent. I n D. vulgaris flavodoxin Trp-60 is inside and Tyr-98 occupies the outside of the flavin ring. The indole and isoalloxazine planes are not parallel in either structure, but Tyr-98 is stacked with the flavin ring in D. vulgaris flavodoxin. All the hydrogen bonds between the flavin ring and the protein appear to be different in the two structures. The only possible similarity would be the interaction of the flavin 0-2 with a backbone N H (89 in Clostridium MP, 95 in D. vulgaris). This bond is not drawn in Fig. 5, because the angular orientation is poor. The N-3 and 0 - 4 interactions utilize dissimilar segments of the polypeptide chain in the two molecules. A backbone NH is the nearest neighbor of the flavin N-5 in both cases, being about 4 A from that flavin atom. While this distance appears too great for formation of a hydrogen bond, the accuracy of the initial models is insufficient to preclude its formation, and Watenpaugh et al. (64) have included this interaction in their hydrogen bonding scheme (Fig. 4b). Burnett et al. (66) concluded that this hydrogen bond is unlikely, both because N-5 in oxidized flavins is not a very good acceptor (96,973 and because the distance between N-5 and the Asp-58 amide is found to be 4.2 A after difference Fourier (83)and real space refinement (85) of oxidized Clostridium M P flavodoxin. In both structures two acidic groups are in the neighborhood of the flavin: residues 58 and 59 in Clostridium M P flavodoxin and 62 and 99 in D. vulgaris flavodoxin. They are oriented somewhat differently in the two molecules. Assuming that electron transfer occurs within a molecular complex between flavodoxin and a “reductase,” one might have expected the three-dimensional arrangement in the neighborhood of the flavin ring to be more highly conserved. The differences ought to be reflected in the relative efficiencies of transfer from the two flavodoxins to a given acceptor. d. Distv-ibution of Residues. The results of chemical modification of cysteine, tyrosine, and tryptophan have suggested that integrity of these side chains is essential for maintenance of the F M N binding site (Section II,C,4). Hence, the position of these residues in each structure is of special interest, In Ctostridium MP flavodoxin, none of the cysteine residues is in direct contact with FMN. The nearest Cys, a t position 53 in the parallel sheet, is adjacent to Ser-54, which forms a hydrogen bond to the flavin phosphate, but the side chain of Cys-53 necessarily protrudes 96. M. Sun and P.4.Song, Biochemistry 12,4663 (1973). 97. F. Miiller, P. Hemmerioh, and A. Ehrenberg, in “Flavins and Flavoproteins” (H. Kamin, ed.), p. 107. Univ. Park Press, Baltimore, Maryland, 1971.

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FLAVODOXINS AND ELECTRON-TRANSFERRING FLAVOPROTEINS

79

from the opposite side of the sheet, away from the FMN. The remaining cysteine sulfurs a t residues 128 and 108 are at distances of 12 and 22 A, respectively, from the F M N phosphorus. The relative positions of the tyrosine and tryptophan residues of Clostridium M P flavodoxin can be seen in Fig. 9. All the tyrosines are separated from the F M N by intervening atoms. Tryptophan-90, on the other hand, is an immediate neighbor of the isoalloxazine ring and would be expected to influence the properties of the protein-bound flavin. The five phenylalanine rings are all inside the molecule, with 66, 69, and 99 forming a central hydrophobic cluster. Desulfovibrio vulgaris flavodoxin contains four cysteines ; as in the Clostridium M P molecule, none of these is involved in a disulfide linkage. Cysteine-57 is in the same position as Cys-53 of the Clostridium M P protein. The backbone amide N of Cys-102 forms a hydrogen bond to the isoalloxazine 0-2, and Cys-93, in the neighborhood of the phosphate, corresponds approximately to Ser-87 of Clostridium M P flavodoxin. However, neither of these -SH groups interacts with atoms of FMN. Cysteine90 of D . vulgaris flavodoxin is more distant from the FMN binding site. Both Tyr-98 and Trp-60 are clearly involved in flavin binding in the D. vulgaris protein (64,78). According to the sequence analysis, there are nine aspartate and 19 glutamate residues in Clostridium M P flavodoxin, but only two arginines and 10 lysines. Four of the acidic groups are clearly paired with basic residues, and many of the remaining carboxylates are within 5-7 A of the charges on arginines or lysines. However, two clusters of uncompensated negative charge occur on the surface of the molecule. One acidic region, residues 62-67, is near the flavin; and the other, residues 120-125, includes the ligands which bind samarium. The arrangement of charged areas may be functionally important since the binding of flavodoxin to ferredoxin-NADP+-reductase is very dependent on ionic strength (42-45). e. Structure as a Function of Oxidation State. Comparisons of independently phased oxidized and semiquinone structures (Table I) have been made for crystalline Clostridium M P flavodoxin (69). The molecular displacement accompanying reduction complicates direct comparison of the two electron density maps. To detect possible conformational changes, small rotations and translations had to be applied to the map of the semiquinone form to maximize its correspondence with the oxidized density (98). The resulting density was then optically superposed on the skeletal model of oxidized flavodoxin. In nearly all regions the agreement between the semiquinone map and the oxidized structure was close. I n particular, the conformation of the ribityl side chain appeared to be unchanged, and the isoalloxazine ring was essentially planar. Real space refinement of 98. J. M.Cox, JMB 28, 151 (1967).

80

STEPHEN G. MAYHEW AND MARTHA L. LUDWIG

111

Ibl

FIO.6. The proposed conformations of residues 58-59 in (a) the oxidized form and (b) the semiquinone form of Clostridium MP flavodoxin. The drawings are based on coordinates obtained by real space refinement (86). The view is not quite perpendicular to the flavin ring. Except for the N-5 proton in the semiquinone, hydrogens have been omitted, nor are the full electronic structures shown Ccf. formula (I), Section II,D,51.

the F M N (69,86)yielded a bending angle similar to that found for oxidized flavodoxin (98~). Several discrepancies between the oxidized model and the semiquinone density have been observed in the FMN binding site and attributed to conformational differences (69).Some of the changes proposed to accompany electron transfer are illustrated in Fig. 6. The density suggests that a movement of the indole ring of Trp-90 and a rearrangement of the bend involving residues 56 through 59 result from one-electron reduction of the FMN. The tryptophan ring motion is complex, involving rotations about both the C,Cp and C,g-C, bonds, but the angle between the indole ring and the mean flavin plane changes only slightly as a result. In the map of oxidized Cbstridium M P flavodoxin, the density suggests that the carbonyl oxygen of Gly-57 points down, away from the FMN, and in nearly the same direction as the p-carbon of Asp-58. The observed conformation resembles the energetically unfavorable Type I1 3,, bend (80). However, in the semiquinone structure the bend appears to revert to the more stable Type I arrangement by a reorientation of the peptide connecting residues 57 and 58, allowing the formation of a hydrogen bond between N-5 and the backbone oxygen 57, which is now about 3.0A from the flavin nitrogen. Hydrogen bonding in the bend may also be perturbed in the semiquinone structure since the peptide planes shift slightly (Fig. 6). Conformational changes as subtle as those suggested by the electron density maps are di5cult to prove by crystallographic methods especially 98a. The bending angles for the flavin ring in reduced and radical forms of Clostridium MP flavodoxin have been found to be about 9" and 6', respectively.

2.

FLAVOWXINS AND ELECTRON-TRANSFERRING FLAVOPROTEINS

81

when accompanied, as in this case, by the small movement of many other atoms. A difference synthesis using terms m(lFI,, - IF,,) exp iaox ( 7 9 ) , with phases determined by isomorphous replacement, contained features consistent with the above interpretation but was not definitive. Therefore, refinement and extension of the data have been undertaken. If the data and phases can be improved sufficiently, then difference Fourier maps with coefficients ( lFlobs- (Flcale) exp iaeslc(79) should reveal the conformation of residues 56-59 when these atoms are omitted from the structure factor calculation. For oxidized flavodoxin, the use of calculated structure factors with R = 0.27 and data to 1.9 A resolution results in densities corresponding to the 0-57 arrangement deduced from the isomorphous replacement maps ( 6 9 ) . Final verification of the proposed changes will require further refinement of the models for both oxidation states. The structural differences indicated by the two isomorphous replacement maps, if proved valid, have several chemical implications. The flavin semiquinone is bound more firmly by the protein than is oxidized F M N ; the change in I(, reflects the perturbation of the redox potentials of F M N by the protein (Sections II,C,2 and E) . Additional FMN-protein interactions and formation of the more stable Type I bend in the semiquinone state would both tend to account for the larger K,, for association of the FMN semiquinone. Undoubtedly other phenomena, such as the altered charge distribution in the flavin ring, also contribute to the net change in free energy of association. Further, the formation of a hydrogen bond between the protonated N-5 and a peptide oxygen provides a means of stabilizing the neutral form of the flavin radical. The pk’ of the N-5 proton is displaced upward by more than two units in the presence of the protein (Section 11,DJ). The ionization of this proton may also be affected by the negative charges on Glu-59 and Asp-58. Finally, if a conformational change must occur during the oxidation-reduction reaction, then the potential energy barrier for rearrangement might limit the rate of electron transfer. It should be emphasized that the proposed structural changes, associated with formation of the semiquinone of Clostridium M P flavodoxin, are unlikely to be duplicated in all flavodoxins. The Clostridium M P and D. vulgaris structures are folded differently in the loops adjacent to the isoalloxazine ring. I n particular, the oxidized D. vulgaris chain does not form a 310bend in the region corresponding approximately to residues 56-59 of Clostridium M P flavodoxin. Thus, although the D. vulgaris protein stabilizes the neutral F M N semiquinone and has an E2 redox p6tential somewhat different from that of free FMN, the structural “explanations” for these phenomena cannot be precisely the same as those offered for Clostridium M P flavodoxin.

82

STEPHEN G. MAYHEW AND MARTHA L. LmwIa

X-Ray intensities for fully reduced crystals of Clostridium M P flavodoxin have been measured to 2.5 A resolution. The intensities are almost identical with those for semiquinone crystals ( R I = 0.06) ; difference Fourier maps comparing the semiquinone and reduced states do not suggest any large rearrangements involving the F M N or its surroundings (69). Other techniques affirm that the conformations of the semiquinone and reduced states of flavodoxins are very similar. Neither the NMR spectra of Clostridium M P flavodoxin (99) (Section II,D,5) nor temperature-jump studies of the reduction of A . vinelandii flavodoxin (100) provides evidence for conformational changes accompanying formation of the fully reduced states. The crystallographic results suggest that the flavin ring in reduced Clostridium M P flavodoxin is nearly planar (98a). This is presumably not its most stable conformation ; structural analyses of fully reduced flavins (89) indicate that the dihydroisoalloxazine ring prefers a conformation which is folded along the N-5:N-lO line.

C. FLAVIN-PROTEIN INTERACTIONS: CHEMICAL AND PHYSICAL STUDIES IN

SOLUTION

1. Preparation and Properties of the Apoprobein FMN is released from flavodoxins by treatment with TCA (15,46,61) or other acids (5,11,59,60), 2 M KBr at pH 3.9 (59), guanidine hydrochloride a t pH 7 (8,11), and, in certain cases, by reaction with mercurials (8,13,40). Solutions of apoflavodoxins are colorless, with a single absorption maximum in the near UV (A, 280 nm, c = 25,000-26,000 M-l cm-l) and fluoresce upon excitation of tyrosyl and tryptophanyl residues (59,62,101). Circular dichroism spectra in the far UV show that the secondary structure of apoflavodoxins is different from that of native flavodoxins (62). In A . vinelandii flavodoxins, this change in conformation on removal of the flavin does not affect the overall exposure of tryptophan residues, but, as judged by the effects of ethylene glycol on the UV absorption spectrum, it may decrease the exposure of tyrosine residues to solvent (101). Nevertheless, the tyrosines titrate normally in the apoprotein whereas in the holoprotein their pK values are displaced upward and the titration becomes partly irreversible (106,103). CI

99. T. L. James, M. L. Ludwig, and M. Cohn, Proc. Nat. Acad. Sci. U. S. 70, 3292 (1973). 100. B. G.Barman and G. Tollin, Biochemistry 11,4755 (1972). 101. J. A. D’Anna, Jr., and G. Tollin, Biochemistry 10,57 (1971). 102. D.E.Edmondson and G. Tollin, BiochemCtry 10, 133 (1971). 103. G. Tollin and D. E. Edmondson, in “Flavins and Flavoproteins” (H. Kamin, ed.), p. 153. Univ. Park Press, Baltimore, Maryland, 1971.

2.

FLAVODOXINS AND ELECTRON-TRANSFERRING

FLAVOPROTEINS

83

Reconstitution of the holoproteins can be achieved with good yields provided precautions are taken to avoid oxidation of sulfhydryl groups, which appear to be more accessible in the apo- than in the holoproteins (59,104).The CD spectra of native and reconstituted proteins are very similar ( 6 2 ) . Complete regeneration of the structure has been demonstrated for Clostridium M P flavodoxin ; after reconstitution this protein yields crystals whose diffraction patterns are identical with crystals of untreated protein (105). 2. Themnodynamics and Kinetics of Flavin Binding

Association constants for FMN and apoflavodoxins from A . vinelandii

(60,61), P. elsdenii (59,104), and other flavodoxins (59,100)have been

determined by equilibrium titrations monitored by fluorescence or absorbance measurements (see Table 111). In the case of the A . vinelandii protein, the constant is almost independent of p H from p H 4.5 to 8.0 (90).Since the second ionization of the F M N phosphate occurs near p H 6 (106) these results imply that both the moao- and dianion forms of the phosphate are bound with approximately equal affinity. Below p H 4.5 the association constant decreases abruptly, suggesting that protonation of two groups affects the FMN-protein interactions (90). The equilibrium constants for association of F M N semiquinone and hydroquinone with apoflavodoxins can be calculated from the association constants of the oxidized protein and the measured shifts of the redox potentials of F M N (59,100). Such calculations show that F MN semiquinone is usually bound very much more tightly than either the oxidized or fully reduced flavin. The calculated K , differences are especially dramatic for flavodoxin from A . vinelandii (Table 111). During F M N binding the protein and flavin fluorescences are quenched in parallel and the quenching reaction appears to be second order (59,61,101). For P. elsdenii flavodoxin a maximum rate of association occurs near pH 4.5, but for A . vinelandii flavodoxin the reaction rate continues to increase as the pH is lowered to about 4 (90).Barman and Tollin ( l o r ) ,employing temperature-jump techniques, have shown that association of F MN with apoflavodoxin from A . vinelandii actually proceeds in two steps. After applying the temperature perturbation, they observed a decrease in flavin fluorescence during about 5 sec followed 104. S. G. Mayhew, in “Flavins and Flavoproteins” (H. Kamin, ed.), p. 185. Univ. Park Press, Baltimore, Maryland, 1971. 105. M. L. Ludwig, R. Andersen, P. A. Apgar, and M. LeQuesne, in ‘(Flavins and Flavoproteins” (H. Kamin, ed.), p. 171. Univ. Park Press, Baltimore, Maryland, 1971. 106. H. Theorell and A. P. Nygaard, Acta Chem. Scand. 5, 1649 (1954).

TABLE I11 BINDINGOF FLAVINS BY FLAVODOXINS' Apoflavodoxin

A. vinelandii Flavin FMN derivatives FMN, oxidized semiquinone reduced ZPropyl%Methyl3-CHzCOO5DeaeaIsoDWXYRiboflavin derivatives Riboflavin DeoxyISO-

K., M-1

(24", pH 7) 2.0 x l o a d 5.8 X lolac 1.4 x 1091

3.7

x x x

ki," M-I sec-' 2

x

106

107

107 4 4.8 107 1 . 3 X 108

4

x

104

1.8 X 1 0 6 2.4 X i o a 1.7 X 106 6.3 x 107

8.9 X

lo6

K . (20", PH 7) 2.3 x 2.9 X 1.1 x 1.3 x 3.3 x 1 x

109 10'' 108 104 108 107

++ 2.3 X 10' -(
klJc(0.5" pH 7) 1.4 X

lo6

6.8 X lo4 3.2 X 108 6 X 10' 4.9

x

104

Radical at half reduction (%) 95

D. vulgaris K., pH 8.2 8.2 x 107 5 X looe 5 x 10k

66 Small

Ref. 13,69, 61,100 65 6a,61

63

107,108

79

63,61

63,61

1.3 X 10''

61,69,107 61 63.61 61.

Riboflavin SO4 Other flavins N-10 w-Carboxvbu t,ylisoalloxkzine 3.1 X 106 63,61 FAD 1 3 x / isA1 ,. in6 ---,-Likiflavin 2.2 x 105 4 x 107 63,61,107 a No entry indicates not measured; indicates compound binds, K . not measured; and - indicates binding not detectable under experimental conditions. !This protein also binds 6- and &OH-FMN, and 7,&dichloro-FMN. For the related Clostridium MP flavodoxin, using K. for oxidwed P. elsdenii flavodoxin and redox potentials at pH 7 (Table IV), K . for the semiquinone = 7.1 X 10" and K,, for the hydroquinone = 1.0 x 108. Overall bimolecular rate constant for formation of the flavin-protein complex. Reported values range from 1.7 to 2.2 X 108. Redox potentials at pH 8.2 were used to calculate these values. f pH 7.

+

?

Eic

u

m

E

e

P

2.

FLAVODOXINS AND ELECTRON-TRANSFERRING

FLAVOPROTEINS

85

by a slower increase during a further 25 sec. The faster relaxation was independent of concentration. They concluded that in F M N binding, the initial second-order reaction of flavin and protein precedes tt faster rearrangement in which fluorescence is further quenched. For A . vinelandii flavodoxin at loo and pH7: Apoprotetn

+ FMN

kl =

5.3

X

k-,= 8.1

X

lo4 M-' sec-' sec-l

(Apoprotein - FMN)

(2)

k, = 0.16 sec-'

a_,= 4.2

X

sec-I

Holoprotein

Because T~ GS r2 a t the concentrations used, the two relaxations had to be resolved by analog fitting to the experimental traces. The forward bimolecular rate constant, k,, is in reasonable agreement with values obtained by stopped-flow measurements (61).Rate constants were not evaluated for P . elsdenii flavodoxin, which displays similar behavior, but the first relaxation appears to be more rapid for this protein. The occurrence of a first-order process is consistent with the shifts in CD spectra resulting from addition of FMN (62),but the observed relaxation should not be ascribed t o changes in the secondary structure of the protein until rates of the CD spectral changes have been determined. Binding of FMN to the apoprotein of A . vinelandii flavodoxin involves a large positive entropy of activation; the activation energy for the reaction is 15.8 kcal/mole (lor),in contrast with the value of 8.3 kcal/mole determined for P . elsdenii flavodoxin (59). 3. Binding of Modified Flavins

The flavin binding site of flavodoxins has been explored by studying the effects of substituting other flavins for FMN. The energies of binding and the properties of the complexes with different flavins vary widely (Table 111).The most extensive studies of the equilibria and kinetics of binding of flavin analogs have been carried out with A . vinelandii flavodoxin by Tollin and co-workers (61,107,108). More limited data are available for the P . elsdenii and D. vulgaris proteins. The free energies and kinetics of binding are affected by substitutions or deletions a t any of a number of positions in the prosthetic group. Deletion of a substituent does not always provide an estimate of its contribution to the overall energy of interaction. Some effects appear explicable only if one assumes 107. B. G. Barman and G. Tollin, Biochemistry 11, 4746 (1972). 108. D. E. Edmondson, B. Barman, and G. Tollin, Biochemistry 11, 1133 (1972).

86

STEPHEN G. MAYHEW AND MARTHA L. LUDWIG

that the conformation of the protein and/or the flavin in analog complexes differs from that found in the FMN holoproteins (61). The effects on K, of substitution in the isoalloxazine moiety are generally consistent with the multiple interactions between the flavin ring and the protein observed in the known structures (Table 111).Substituents at N-3 and C-2 uniformly decrease the association constants. Although the dimethylbenzene end of the flavin ring is relatively accessible to solvent, the introduction of a methyl group a t position 6 (iso-FMN) can be shown by model building to produce close contacts with residue 57 in Clostridium M P flavodoxin. The redox properties of FMN derivatives of A . vinelandii flavodoxin modified a t positions 2, 3, and 6 differ from those of native flavodoxin; the potentials and rates of reduction Nevertheless, in many derivby dithionite are significantly altered (102). atives of A . vinelandii or P. elsdenii flavodoxin a semiquinone form is stabilized a t half-reduction (102) and biological electron transfer reactions can proceed, albeit with diminished efficiency, when modified flavins are incorporated into P. elsdenii flavodoxin (63).Deaza-FMN is isosteric with F M N and its different behavior must be attributed to altered electronic properties (96,108). Bound deaza-FMN does not form a semiquinone, but is instead partially converted to the fully reduced form by reaction with dithionite (108). From the effects of this analog on the rates and equilibria of binding, an interaction between the protein and N-5 of the F M N in oxidized A . vinelandii flavodoxin has been inferred (108). Such an interaction is not predicted from the structure of Clostridium M P flavodoxin (Section II,B,3). The results of modification of the ribityl side chain vary with the species of flavodoxin. The hydrogen bonding interactions of OH-2’ and OH-4’, observed in the structure of Clostridium M P flavodoxin, would be expected to favor association. Removal of these groups ought to reduce K,. Assuming isomorphism of the Clostridium M P and P. elsdenii structures (109), comparison of the constants for deoxy-FMN and FMN binding to P . elsdenii flavodoxin suggests that these interactions contribute approximately 4 kcal of binding energy. Similar contributions for the D . vulgaris protein are predicted by its structure. Surprisingly, the constants for F M N and deoxy-FMN are reported to be almost identical for A . vinelandii flavodoxin ; however, there must be some stereochemical restrictions on the conformation of the ribityl side chain, since tetra-0109. Because of their similar properties and the homology of the sequences constituting their active sites, Clostridium MP and P . etsdenii flavodoxins have been considered equivalent when the chemical results are compared with the structural data. The term “clostridial flavodoxin” is used to include both C. pasteurianum and Clostridiurn MP flavodoxins.

2.

FLAVODOXINS AND ELECTRON-TRANSFERRING FLAVOPROTEINS

87

acetyl riboflavin fails to bind to the A . vinelandii apoprotein (61). Derivatives with four or six carbon atoms in the side chain do not bind to P. elsdenii apoflavodoxin ( 11 0 ) . The role of the phosphate group in stabilizing the F M N complexes is most intriguing. The apoflavodoxins from A . vinelandii, R. rubrum, and D. vuEgaris all bind riboflavin. I n contrast, apoflavodoxins from Clostm'diu and P. elsdenii are specific for flavin a t the level of F MN (59). Even though the latter proteins have higher affinities for F M N than do the D. vulgaris or A . vinelandii proteins, they do not bind riboflavin (or lumiflavin or FAD) to a detectable extent ( K , < lo3),These observations do not necessarily imply that ring interactions contribute little to the binding energy. Nervertheless, the apparent contribution of the phosphate-protein interactions is very much greater for the clostridial and P. elsdenii flavodoxins than for A . vinelandii or D. vulgaris flavodoxins. Yet the structures show these interactions to be essentially identical in the Clostridium M P and D. vulgaris proteins. To resolve this dilemma, differences in the apoprotein structures have been invoked. From the biphasic binding kinetics and other observations it has been postulated that the ,iif phosphate of FMN is necessary to trigger a conformational D'Anna and Tollin ( 6 2 ) have prochange in the apoprotein (62,107). posed that with the FMN-specific proteins this conformational change is much larger. In support of this suggestion, they showed that when flavin is bound by the FMN-specific apoproteins, there is a relatively large change in the ellipticities of the C D bands in the f a r UV region (WS,62). On the other hand, the kinetics of riboflavin binding to A . vinelandii apoprotein show no evidence of a structural rearrangement (10'7). Conceivably, then, the structures of some of the complexes with dephospho analogs are different from those of the FMN proteins. It may be significant that complexes of deoxyriboflavin and N-10-o-carboxybutyl isoalloxazine with A . vinelandii apoflavodoxin fluoresce and differ in other respects from the native flavodoxin and that the riboflavin complex forms negligible amounts of semiquinone (61). Unfortunately, the far UV circular dichroism spectra of riboflavin and related complexes have not been reported. The crystal structures of suitable analog complexes would also be of great assistance in evaluating some of these data. 4. Protein Modifications That AffectFlavin Binding

Chemical modification of cysteines seemed to implicate these residues in maintenance of the flavin binding site of certain flavodoxins. Knight and Hardy (8) found that F M N dissociated from C. pasteurianum flavodoxin upon reaction with sodium mersalyl and proposed that the single 110. R. Gast and F. Miiller, private communication.

88

STEPHEN G. MAYHEW AND MARTHA L. LUDWIG

sulfhydryl group in this protein might be important for flavin binding. Subsequent work with Clostridium M P (59), P. elsdenii (59), and D. vulgaris (40) flavodoxins supported the view that one or more sulfhydryls were involved in flavin binding in these proteins. On the other hand, flavin binding by A. vinelandii flavodoxin is not prevented by reaction of the apoprotein with DTNB (111). The possibility that -SH groups bind directly to flavin atoms in Clostridium M P and D. vulgaris flavodoxins has been ruled out by the two structures (Section II,B,3) and it must be concluded that the observed inhibition of F M N binding by N-ethylmaleimide and mercurials (59,104) arises indirectly from effects on the secondary or tertiary structure of these proteins. Chemical evidence for the proximity of tyrosines to FMN in A. vinelandii and P. elsdenii flavodoxins is based on modification by tetranitromethane. Hinkson (60) observed that nitration of four of the five tryosines in A . vinelandii apoprotein eliminated most of its capacity to bind FMN and that in the native protein two tyrosines are protected against nitration. Flavin binding by P . elsdenii apoflavodoxin is similarly inhibited after reaction with TNM (59) and also by reaction with 5 moles of iodine (118).The two -SH groups were protected during iodination and consequently a modification of tyrosines seemed likely. I n the absence of a structure, conclusions about the interaction of tyrosine and F M N in A. vinelandii flavodoxin cannot be assessed, but assuming the similarity of P. elsdenii and Clostridium M P flavodoxins (109) then tyrosyl modifications must exert an indirect effect on flavin binding in both of these proteins since no tyrosines are in contact with FMN. On the basis of results of experiments with N-bromosuccinimide, McCormick (113) proposed that a tryptophan might be complexed with the flavin of C. pasteurianum flavodoxin, a suggestion consistent with the known structures. Ryan and Tollin (111) conducted similar experiments with A. vinelandii flavodoxin, concluding that reaction of a single tryptophan was responsible for loss of F M N binding capacity.

D. SPECTROSCOPIC PROPERTIES 1. Optical Absorption Spectra of Solutions

In oxidized flavodoxins the bound flavin exhibits two broad absorption bands in the visible (Fig. 7) and a third band in the near UV (A,,, 272-275), overlapping the aromatic absorbances of the protein. The peak 111. J. Ryan and G . Tollin, Biochemistry 12, 4550 (1973). 112. E. A. Gowie and S.G. Mayhew, unpublished. 113. D. B. McCormick, Experientia 15, 243 (1970).

2.

FLAVODOXINS AND ELECTRON-TRANSFERRING I

I

I

I

I

89

FLAVOPROTEINS I

I

I

I

Wovelength (nm)

FIO.7. Absorption spectra of flavodoxins in solution. Curves 1 and 3: oxidized and semiquinone forms, respectively, of A . vinelaiidii flavodoxin; curves 2, 4, and 5 : oxidized, semiquinone, and fully reduced forms, respectively, of P. elsdenii flavodoxin.

positions, shapes, and intensities of the visible bands vary considerably with the source of the protein and differ from those of free F M N (Table IV) ; for example, the maximum of F M N a t 445 nm is red-shifted by 5-22 nm in most flavodoxins, slightly blue-shifted to 443 nm in flavodoxin from C . pasteurianum, but unchanged in flavodoxins from P . elsdenii and Clostridium MP. The extinction coefficient a t this maximum is usually about 85% of that of free FMN, and in addition the band has more or less pronounced shoulders on each side of the maximum as a result of vibronic splittings. The position and structure of the 373-nm band of F M N is likewise altered in the proteins. Temperature difference spectra of several flavodoxins reveal the vibrational structure of the two visible bands in more detail (114). Peak shifts and vibrational splittings are commonly observed in other flavoproteins and also occur with model flavins when the polariaability or hydrogen bonding capacity of the solvent is varied or when intermolecular complexes are formed with compounds such as indoles (116,116).The inference that in flavodoxins the 114. F. Miiller, S. G . Mayhew, and V. Massey, Biochemistry 12, 4854 (1973). 115. H. A. Harbury, K. F. LaNoue, P. A. Loach, and R. M. Amick, Proc. Nut. Acad. Sci. U.S . 45, 1708 (1959). 118. G. Weber, BJ 47, 114 (1950).

90

STEPHEN G. MAYHEW AND MARTHA L. LUDWIG

flavin lies in a relatively nonpolar environment, perhaps containing aromatic residues, is borne out by the structures of the Clostridium M P and D . vulgaris proteins, and the considerable differences in the visible spectra of these two flavodoxins (Table IV) may be attributed to the more aromatic environment of the flavin chromophore in the D. vulgaris molecule. Upon addition of one reducing equivalent, free isoalloxazines are converted to mixtures of the three oxidation states in which the semiquinone constitutes a minor fraction (117). Because of the shifts in redox potential (Table IV) the distribution a t equilibrium in flavodoxin solutions favors the semiquinone in yields approaching 100% a t neutral pH (118). Flavodoxin radicals always display the blue spectrum assigned to the neutral species with a proton a t N-5 (118-121) [formula (I), Section II,D,51; they are not converted to the red anionic form even at pH values above 10 (12,13,102), although the pR of the free FMN radical is approximately 8.6 (117,121).The absorption spectrum of the semiquinone of P . elsdenii flavodoxin (Fig. 7) has maxima at 350, 377, 505, and 580 nm, and a broad shoulder at 627 nm. Spectra of semiquinones of clostridial flavodoxins are similar (7,lS) ; other flavodoxins absorb less around 500 nm (14,16,23,51); and in A . vinelandii flavodoxin the long wavelength shoulder becomes a peak in the region of 615 nm ( 2 3 ) . Muller et al. (122) have proposed that spectra of the latter type indicate less accessibility of the flavin radical to water. Edmondson and Tollin (102) found that a number of flavodoxin semiquinones show a large decrease of absorption around 285 nm, and an increase around 270 nm, compared with the absorption of the oxidized protein. The decrease a t 285 corresponds to a similar change in the spectrum of free FMN on reduction to the semiquinone (102,123); it is suggested that the increase a t 270 nm is either the result of a red shift of the UV absorption maximum of the semiquinone, which lies below 260 nm in free FMN semiquinone (123), or of a charge transfer interaction between the semiquinone and an aromatic amino acid. 117. A. Ehrenberg, F. Muller, and P. Hemmerich, Eur. J . Biochem. 2, 286 (1967). 118. V. Massey and G. Palmer, Biochemistry 5,3181 (1966). 119. G. Palmer and V. Massey, in “Biological Oxidations” (T. P. Singer, ed.), p. 263. Wiley, New York, 1968. 120. G. Palmer, F. Muller, and V. Massey, in “Flavins and Flavoproteins” (H. Kamin, ed.), p. 123. Univ. Park Press, Baltimore, Maryland, 1971. 121. F. Miiller, P. Hemmerich, A. Ehrenberg, G. Palmer, and V. Massey, Eur. J. Biochem. 14, 185 (1970). 122. F. Miiller, M. Briistlein, P. Hemmerich, V. Massey, and W. Walker, Eur. J . Biochem. W, 673 (1972). 123. E. J. Land and A. J. Swallow, Biochemistry 8, 2117 (1969).

2.

FLAVODOXINS AND ELECTRON-TRANSFERRING FLAVOPROTEINS

91

Fully reduced flavodoxins from P. elsdenii and Clostridia are pale yellow with Amax near 365 and 315 nm and a broad shoulder centered around 450 nm (Fig. 7). Resemblances to the low temperature spectrum of the anionic form of reduced tetraacetyl-riboflavin suggest that the reduced protein-bound flavin is the anion, and the presence of the long wavelength absorbance appears consistent with a planar conformation for the reduced isoalloxazine in these proteins (1244).Unfortunately, there is little similarly detailed information about the spectroscopic properties of other flavodoxin hydroquinones. However, some differences from P. elsdenii flavodoxin seem likely; for example, the hydroquinone of A. nidulans flavodoxin appears to have less absorbance a t 450 nm (125) and flavodoxin from Chlorella fusca has a peak a t 395 nm rather than near 370 nm (15). 2. Optical Absorption Spectra of Single Crystals

Single crystal spectroscopy offers a means of comparing dissolved and crystalline states. An isotropic ‘‘solution” spectrum can be reconstructed by appropriate averaging of the spectra obtained when a crystal is oriented so that incident light is polarized either parallel or perpendicular to selected crystal axes. Calculation of the flavin semiquinone spectrum from measurements on crystals of Clostridium M P flavodoxin was of special interest since the reddish color of these crystals, noted on examination in unpolarized light (105), suggested that changes in the flavin spectrum might have occurred as a result of crystallization. Spectra of single crystals of Clostridium MP flavodoxin are reproduced in Fig. 8 along with the calculated isotropic spectra (126). The reconstructed semiquinone spectrum is typical of the blue neutral flavin radical. Hence, the radical does not undergo tautomerization or conversion to the anionic form upon crystallization. These conclusions from optical spectroscopy are supported by measurements of the linewidth (19 G) of the E P R spectrum of a crystalline powder sample of flavodoxin semiquinone (Section

II,D,5).

From the optical measurements on crystals of Clostridium RiIP flavodoxin (Fig. 8) and the known orientation of the isoalloxazine ring with respect to the crystal axes, i t has also been possible to assign the transition moment directions of the r-r* transitions responsible for the absorbance of the oxidized flavin a t 376 and 445 nm (13,126). Components 124. S. Ghisla, V. Massey, J.-M. Lhoste, and S. G. Mayhew, Biochemistry 13, 589 (1974). 125. B. Entsch and R. M. Smillie, A B B 151, 378 (1972). 126. W. A. Eaton, J. Hofrichter, M. W. Makinen, R. D. Andersen, and M. L. Ludwig, Biochemistry 14, 2146 (1975).

92

STEPHEN G. MAYHEW AND MARTHA L. LUDWIG

350

WAVELENGTH (nml 4w

WAVELENGTH (nml

500

'

L a

* I

Ibl

la)

It1

Fro. 8. Single crystal, solution, and polarization ratio spectra of flavodoxin from Clostridium MP. The crystal spectra were measured with the electric vector of the polarized light parallel and perpendicular to the c axis. The polarization ratio, P , is defined as qc/eIc, and is related to 6,the angle between the transition moment directions and the c axis of the crystal. (a) Oxidized flavodoxin. The isotropic spectrum was calculated assuming equal extinction coefficients of 10.4 mM-1 cm-1 for the peaks of the solution and isotropic crystal spectra near 22,000 cm-I (13). (b) Flavodoxin semiquinone. The calculated isotropic spectrum assumed the extinction coefficient of 4.6 mM-1 om-' a t 17,250 cm-I (13). (c) Possible transition moment directions for the a (445 nm) and @ (376 nm) transitions of the oxidized form of Clostridium MP flavodoxin. The angles are defined relative to the y axis, which lies in the flavin plane and is perpendicular to the line joining N-5 and N-10. Directions determined experimentally are the solid arrows, while the dotted arrows are the directions predicted from theory (167). The values of (I = 15" and @ = -5' are considered to be the correct assignments. From Eaton et al. (166).

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FLAVODOXINS AND ELECTRON-TRANSFERRING FLAVOPROTEINS

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of these moments parallel and perpendicular to the c axis are derived from the polarization ratios Ell/4,. Loci of the transition moment directions then describe cones about c, whose intersections with the isoalloxazine plane represent the two permissible directions for each in-plane transition. The polarized emission spectra of model flavins, measured b y Sun et al. (127) suggest that the angle between the two transition directions must be about 20°, and measurements on stretched films (128)indicate that both transitions are nearly parallel to the long axis of the isoalloxazine system. This information further restricts the assignment of the moments. The directions shown in Fig. 8c are in good agreement with theoretical calculations for model oxidized isoalloxazines (127). The low energy transitions for the semiquinone, centered near 580 and 500 nm, have directions very similar to those for the 445- and 376-nm transitions of the oxidized form. As a result it has been postulated that the *-orbital charge density is very similar in the oxidized and radical states of the protein-bound flavin (126). Electron transfer involving these oxidation states would therefore require no gross rearrangement of the electronic structure of the flavin. 3. Fluorescence

The greenish yellow fluorescence of unbound oxidized FMN is quenched in the flavodoxins. The residual fluorescence observed in solutions of several flavodoxins is less than 1% of the fluorescence of F M N (69) and is probably the result of traces of flavin dissociated from the holoprotein; a M solution of a flavodoxin with an association constant of lo@ M-l, as is typical of these proteins, would contain about lo-' M free FMN. For all practical purposes these flavoproteins are nonfluorescent. In D. vulgaris and Clostridium MP flavodoxins, the quenching may be attributed to the presence of neighboring aromatic residues. Though the precise mechanism is unclear, quenching of FMN fluorescence by complex formation with aromatic compounds has been demonstrated in various model systems (129). In contrast to a widely held view, ffavin hydroquinones are intrinsically fluorescent ( l Z 4 ) . In some reduced flavoproteins, this emission is quenched, but in flavodoxin from P. elsdenii a weak fluorescence is observed with a peak a t 530 nm (19.4). The fluorescence of the aromatic residues of apoflavodoxins from C h tridia, P. elsdenii, and D. vulgaris is about 99% quenched upon addition of FMN (69,62). I n flavodoxins from A . vinelandii and R. m b r u m , only 127. M. Sun, T.A. Moore, and P.-S. Song,JACS94, 1730 (1972). 128. J.-M. Lhoste, Proc. Eur. Biophys. Congr., I s t , 4, 221 (1971). 129. R.E.MacKenzie, W. Fory, and D. B. McCormick, Biochemhstry 8,1839 (1969).

94

STEPHEN G. MAYHEW AND MARTHA L. LUDWIG

FIG.9. A stereo view showing the relationship of the tyrosine, tryptophan, and FMN rings in Clostridium M P flavodoxin. From Burnett et al. (66).

partial quenching of the aromatic fluorescence occurs ; the holoproteins are, respectively, 13 and 60% as fluorescent as the apoproteins (62,101). D'Anna and Tollin (101) have suggested that the quenching of the protein fluorescence may result from two effects: a direct molecular overlap of the side chains of some aromatic amino acids and the flavin, and Forster energy transfer between the different aromatic systems in the molecule. The first of these suggestions is confirmed in the X-ray structures of two flavodoxins. Resonance transfer from tyrosine to tryptophan and from tryptophan or tyrosine to FMN (130) may explain quenching of the emission of those residues more distant from the flavin in Clostridium M P flavodoxin (Fig. 9 ) . Measurements of the fluorescence of the tryptophans in A . vinelandii flavodoxin support the interpretation that quenching is dependent on a single tryptophan, probably located in the vicinity of the F M N (111,131). 4. Circular Dichroism

The measured far ultraviolet CD spectra of the flavodoxins, despite some differences in detail, all provide evidence for the occurrence of highly ordered secondary structure similar to that observed in the Clostridium M P and D. vulgaris molecules (62). In the visible region of the spectrum, free F M N has rather weak circular dichroism; the bands become much more intense in the flavodoxins, and 130. P.S. Song, T. A. Moore, and W. E. Curtin, 2.Naturforsch. B.27, 1011 (1972). 131. L. Andrews, M. L. MacKnight, J. Ryan, and G. Tollin, BBRC 55, 1165 (1973).

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their signs are reversed (14,23,62). The C D spectra of six flavodoxins have in common a positive band a t about 470 nm and a more intense negative band around 370 nm. There are qualitative and quantitative differences between the several flavodoxins, however, and these are particularly marked in the region 380-450 nm. By manual fitting of gaussian curves, Edmondson and Tollin resolved the C D spectra and visible absorption spectra into six vibronic bands, three being associated with each of the two electronic transitions of FMN ( 2 3 ) . [Other data (114,132) suggest the presence of at least seven bands in the visible region.] One C D band, a t about 450 nm, is positive in P. elsdenii and clostridial flavodoxins, but negative in flavodoxins from A . vinelandii, R. rubrum, and D. vulgaris. The distinctive C D spectra and variations in the resolution of the visible spectra (Table IV and Fig. 7) prompted D’Anna and Tollin to divide these flavodoxins into two groups (62). Their division correlated with the ability to bind riboflavin, and additional correlations were proposed. The spectral properties seemed to indicate a more apolar environment, possibly involving more aromatic residues, for the FMN in flavodoxins from D. vulgaris, R. rubrum, and A . vinelandii. Now that the structures of representative flavodoxins from each class appear to verify these predictions, it is tempting to speculate that the FMN binding sites of A . vinelandii and R. rubrum flavodoxins bear a closer resemblance to D. vulyaris than to Clostridium M P flavodoxin. 5. Magnetic Resonance Spectroscopy

Because of their low molecular weights, the ability to exchange the prosthetic group, and the convenience of microorganisms as a source for isotopically substituted proteins, flavodoxins have been favorite subjects for magnetic resonance experiments. a. EPR and ENDOR Spectra of Semiquinones. The electronic structures of model flavin semiquinones have been established by a combination of E P R and optical spectroscopy, utilizing isotopic substitution and alkylated derivatives (117,121). Comparison of the magnetic hyperfine interactions in EPR spectra of flavins variously substituted with 15N or 2H has permitted determination of the spin density distribution in the isoalloxazine nucleus (121). Negligible spin density is found in the pyrimidine ring (positions 1 to 4) ; for the neutral radical, the maximum spin density occurs at N-5, followed in decreasing order by N-10, C-8, and C-6. The magnitude of the coupling constant (about 7.5 G ) for an exchangeable proton in neutral flavosemiquinones leads to assignment of formula (I),with the dissociable proton of pK 8.5 a t N-5 (191).

-

132. G. Scola-Nagelschneider and P. Hemmerich, 2. Naturforsch. B 27, 1044 (1972).

96

STEPHEN 0. MAYHEW AND MARTHA L. LUDWIG

H

O

(1)

In proteins the detailed structure of the EPR spectrum disappears as a result of the slow tumbling of the protein-bound radical. Nevertheless, the coupling of the N-5 proton is sufficiently large to alter the line width of the spectral envelope ( l a 0 ) .Thus, the spectra of neutral semiquinones, such as the flavodoxins, typically have a line width of 19 G, which decreases to 15 G upon exchange with ‘H20 (120).The EPR line widths correlate precisely with the optical spectra in distinguishing neutral from anionic protein semiquinones. The EPR spectrum of fully deuterated flavodoxin from S. lividus in ‘H,O contains some hyperfine structure which allows estimation of the coupling to N-5 and N-10 (133-135). Recently, ENDOR spectroscopy of flavodoxins from P. elsdenii and A . vinelandii has yielded additional information on the electronic structure and geometry of the protein-bound F M N radical. ENDOR signals demonstrate the presence of spin density a t positions C-8, C-6, and N-10 Furthermore, the magnitude of the in flavodoxin semiquinones (136,137). hyperfine coupling to CH, (8), which can be correlated with the ionization state of flavin radicals (137),is consistent with the presence of the neutral radical (1) in A . vinelandii flavodoxin. A signal corresponding to the interaction of the spin density a t N-5 and the N-5 proton was not detected (137).Nevertheless, the ENDOR results, in combination with the EPR spectra of deuterated flavodoxins, indicate that the spin density distribution in flavodoxin radicals is similar to that found in model flavin semiquinones. ENDOR spectra of several flavoproteins haZle been interpreted as signifying that the flavin radical adopts a planar conformation (137). b. N M R Spectra. The NMR spectra of flavodoxins from C . pasteurianum (I%), S. lividus (18), Clostridium MP, and P. elsdenii (99) have 133. H. L. Crespi, J. R. Norris, and J . J . Katz, BBA 253, 509 (1971). 134. H. L. Crespi, J. R. Norris, J. P. Bays, and J . J . Katz, Ann. N . Y . Aead. Sci. 222, 800 (1973). 135. J. S. Hyde, L. E. G. Eriksson, and A. Ehrenberg, BBA 222, 688 (1970). 136. J. Fritz, F. Muller, and S. G. Mayhew, Helv. Chim. Acta 56, 2250 (1973). 137. L. E. G. Eriksson and A. Ehrenberg, BBA 293,57 (1973). 138. C. C. MacDonald and W. D. Phillips, in “Fine Structure of Proteins and Nucleic Acids” (G. D. Fasman and S. N. Timasheff, eds.), p. 1. Dekker, New York, 1971.

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been reported; for the latter two proteins, spectra of all oxidation states have been recorded. The spectra of the diamagnetic oxidized and fully reduced forms are nearly identical for either Clostridium MP or P. elsdenii flavodoxins, and therefore, for each of these flavodoxins, the conformation cannot be appreciably changed by two-electron reduction. I n the semiquinone state, certain resonances are selectively broadened by the proximity of the flavin paramagnet. However, the general resemblance of the spectra of the radical and diamagnetic forms, along with the X-ray results, argues against any large conformational changes associated with formation of the semiquinone. I n mixtures of partially reduced forms, the spectra appear additive. This observation permits a limit of <50 sec-I to be placed on kerchange, in agreement with values for rates of cornproportionation and disproportionation established by kinetic studies (Section II,F,l). Only certain aromatic resonances and several lines shifted upfield from the aliphatic region are sufficiently well resolved in spectra of P. elsdenii and Clostridium MP fiavodoxins to permit their assignment to individual residues (99). Some of these resonances are not only ring-shifted but also must represent protons in the neighborhood of the flavin ring since they are absent from the spectrum of the semiquinone form; for example, aromatic proton peaks observed at 5.95 and 6.39 ppm in the spectrum of Clostridium MP flavodoxin are upfield from the shifts expected for tyrosine or tryptophan protons and disappear in the presence of the radical. These are likely to be protons of Trp-90. The FMN proton resonances cannot be identified unambiguously in these spectra. Selective proton labeling of deuterated flavodoxin from S. lividus has afforded simplified spectra which are amenable to more detailed interpretation (134) ; for example, these spectra furnish evidence for the presence of two alternative conformations for a leucine residue. Two NMR studies have been specifically concerned with the environment of the protein-bound FMN. Using fully deuterated apoflavodoxin from S. lividus, reconstituted by addition of ['HIFMN, Crespi and coworkers obtained the spectrum of bound oxidized FMN (18). Not all of the peaks could be assigned unequivocally. Changes in the chemical shifts of FMN protons were attributed to the presence of an aromatic residue stacked parallel to the isoalloxazine ring (18,134). The line widths of the ribityl protons and of one aromatic proton were consistent with rigid attachment to the protein, i.e., with the rotational correlation time predicted for flavodoxin (18). The authors ascribed the narrower resonances of the methyl protons and the remaining benzene proton to effects other than rapid motion of the isoalloxazine system relative to the protein, although some motion of the flavin ring is not excluded (134). The

98

STEPHEN G. MAYHEW AND MARTHA L. LUDWIG

structural results (Section II,B,3) similarly suggest restrictions on the mobility of the protein-bound flavin. Palmer and Mildvan (139)observed a dramatic effect of the flavin radical of P. elsdenii flavodoxin on the relaxation rate of water protons, denoting exposure of the radical to solvent. I n the crystal structures, the dimethylbenzene end of the isoalloxazine ring is clearly accessible to solvent and several peaks of density near the FMN ring have been interpreted as representing bound solvent molecules (64,667.

E. OXIDATION-REDUCTION POTENTIALS Oxidation-reduction potentials for several flavodoxins are included in Table IV. I n every case, the two one-electron steps prove to be well resolved. The magnitude of the potential difference, E , - El,varies from 0.17 to 0.545 V, depending on the pH and the species of flavodoxin, but E, (oxidized-semiquinone) is always more positive than El (semiquinone-reduced) (140). This behavior distinguishes the protein-bound flavin from free FMN, whose potentials, E , = -0.238 V and El= -0.172 V a t pH 6.95 ( l d l ) , imply that the equilibrium for comproportionation FH2-

+ F H + H+ + 2FH2’

(3)

lies far to the left. In contrast, addition of one reducing equivalent to the flavodoxins produces predominantly the semiquinone. The shifts of potential for the protein-bound flavin, relative to FMN, facilitate measurement of the individual Emidpoint values for the flavodoxins since only two oxidation states are observed during most of the titration. For P . elsdenii flavodoxin, E , was originally determined by titration with NADPH in the presence of catalytic amounts of ferredoxin-NADP+-reductase, using indigodisulfonate as an indicator ; Elwas calculated from the concentration of semiquinone and hydroquinone in equilibrium with excess NADPH in the presence of reductase, or by equilibration with hydrogen and hydrogenase (13,58). Similar procedures, utilizing various redox indicators, as well as potentiometric titrations, have been employed with the other flavodoxins (references in Table IV) . The pH dependence of the redox potentials has been examined, over 139. G. Palmer and A. S. Mildvan, in “Structure and Function of Oxidation Reduc-

tion Enzymes” (A. Akeson and A. Ehrenberg, eds.), p. 385. Pergamon, Oxford, 1972. 140. The potential for the semiquinone-reduced couple in flavodoxins is designated El to be consistent with the assignments in FMN, where El is the higher potential but refers to the semiquinone-reduced couple. 141. R. D. Draper and L. L. Ingraham, ABB 125, 802 (1968).

TABLE IV DATAFOR FLAVODOXINS Redox potentials

MW of

Source

holoprotein

C . pasteurianum

14,600

Absorption maxima nm (mM-1 cm-1) 272(40) 272(45.8)

372(7.9) 374(8.47)

443(9.1) 443(10.4)

P . elsdenii

15,0005

272 (47.6)

377(8.75)

445(10.2)

Clostridium M P

1.5,800a

272(46.8)

376(9.1)

445(10.4)

pH

7 8 7 8 7

8

D. vulgaris D. gigas R. rubrum A . vinelandii

16, 300a 16,000 22,800 23,000

E. coli A . nidulans

14,500 20,300

S. lividus C . fusca FMN

17,000 22,000

273(48) 273(47) 272(54.2)

375.5(8.7) 374(8.2) 376(11.3) 371(9.5)

456.5(10.7) 456.5(10.2) 460(11.2) 450(10.6)

274(50) 275

370(6.6)

377

467(8.25) 465(9.2)

270

377

465 (10.2)

275(54.6) 266(31.8)

379(9) 373(10.4)

464(10 rt 0.2) 445( 12.5)

7.8 -

8.2 7.7 7.7

7

8 -

7 -

7

Ei (mV)

-419 -429 -372 - 375 - 399 -408 -440 -

-495 -464 -410 -447 -450 -

-450 - 172

Ef

(mV)

- 132 - 192 -115 - 175 - 92 - I52 - 150 -

-

+50

References 6-8 13 12,13,58 13

11,100 11

14

22,61,100

-270 -240 -221 -281 - 50 -

-238

144

16 &,IS5

51 17,18,1S1 15 141

~

Calculated from the amino acid sequence. The remaining values are determined from amino acid compositions (27).

co co

100

STEPHEN G. MAYHEW AND MARTHA L. LUDWIG

a limited range of pH, for P . elsdenii (68),C. pasteuhnum (1S11@), Clostridium M P (IS), and A. nidulans (186) flavodoxins. The results are consistent with the formulation of the reactions at pH 8 as

where the oxidieed and semiquinone flavins are neutral and the reduced form exists as the anion. As expected from the failure to observe any ionization of the oxidized or semiquinone FMN in P. elsdenii flavodoxin over the pH range 5.8 to 9.2 by spectroscopic techniques, the slope of the line relating E , to pH is -0.059 V in accord with the addition of a proton and an electron in reaction (4). Values of E, are independent of pH in the high pH region but become pH dependent at the lower extreme (Fig. 10) (143).The change in slope corresponds to the titration of a redox linked group with a pK of 5.8 in fully reduced P. elsdenii flavodoxin (68) and a pK of 6.7 in C. pasteurianum and Clostridium M P flavodoxins ( I S ) . These pK values have been identified with the formation of the hydroquinone anion ; in model flavins, this ionization occurs a t N-1 with a pK of 6.7 (181,146). A similar ionization, with pK = 7.0, has been proposed for A . vinelandii flavodoxin (B), implying that the pH dependence of El for this flavodoxin is probably similar to those shown in Fig. 10 (143). Because of the dependence of E2on pH, the equilibrium constant for comproportionation changes with pH; for example, a t pH 6.7 the semiquinone formation constant [K = (FH2')2/(FH)(FH2- FHa)] for P . elsdenii flavodoxin is 43,000,while a t pH 9.3 it decreases to 110 (b8). As noted in Table I11 (Section II,C,%), changes in the reduction potentials of FMN upon combination with the apoprotein require that the association constants for FMN binding vary with oxidation state. Thus, the effect of the protein on the redox potentials can equally well be described in terms of the variable affinity of the protein for each redox species of FMN. The available structural and spectroscopic data have led to some hypotheses regarding the molecular basis for the shifts in

+

142. The redox potential first reported for this protein ( 8) was based on extinction measurements at 443 nm which were assumed to change linearly during reduction; the changes are not linear because of semiquinone formation and the calculation was therefore in error. 143. Failure to consider the different effects of pH on Eland E, has led to inaccurate comparisons of the redox potentials of flavodoxins from different sources (18,100,14464).

144. D. C. Yoch, BBRC 49, 336 (1972). 145. H. J. Lowe and W. M. Clark, JBC 221, 983 (1956).

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-0.3

-0.4 -

I

I

5

I

6

I

7

PH

1

a

I

9

FIG.10. Effect of pH on the redox potentials of flavodoxins. (I, 0 ) CEostridium MP flavodoxin, (E, 0 ) C. pasteurianunz flavodoxin, and (0, 0, A ) P . elsdenii

flavodoxin. From Mayhew (13).

K , (69). Conformational changes accompanying reduction to the semiquinone state, suggested by electron density maps of Clostridium M P flavodoxin, are consistent with the tighter binding of F M N semiquinone. An additional hydrogen bond between F M N and the protein and the rearrangement of atoms in the bend 56-59 to produce a lower energy conformation would both act to increase the relative stability of the semiquinone complex. However, the existence of these conformational changes remains to be conclusively established and they are unlikely to occur in all flavodoxins (Section II,B,3,e). The oxidized-semiquinone potential, E,, is highly dependent on the source of the flavodoxin and does not seem to correlate well with the classification of flavodoxins according to their spectral properties (Table IV). Hence, examination of the structures of the semiquinone state of D. vulgaris or other flavodoxins may reveal additional mechanisms for stabilization of the semiquinone form. The strength of nonbonded interactions between isoalloxazine and the adj acent tryptophan, tyrosine, or methionine residues may also vary with

102

STEPHEN G. MAYHEW AND MARTHA L. LUDWIG

oxidation state. Draper and Ingraham (146) found that the extent of complex formation between riboflavin and tryptophan, tyrosine, or glutathione decreases in the order semiquinone > oxidized > fully reduced. The available X-ray data for fully reduced Clostridium M P flavodoxin indicate that the isoalloxazine ring is nearly planar in the reduced protein (69). The spectra of reduced flavodoxins from Clostridia and P. elsdenii also favor the existence of a planar ring, and the fluorescence of reduced flavodoxins is compatible with the restriction of the bending modes typical of reduced isoalloxazines (124). Free reduced flavins, on the other hand, are bent along the N-5:N-lO direction, with the extent of bending dependent on the nature of ring substituents (89),and they undergo rapid ring inversion (147). It is reasonable to suppose that the decrease in K , for the reduced state results in part from constraints imposed by the protein on the conformation and mobility of the dihydroisoalloxazine ring. The relative destabilization of the reduced FMN-protein complex is presumably the physiologically most significant thermodynamic role of the protein since its effect is to lower E l to the range of potentials characteristic of the ferredoxins ( 1 0 0 ~ ) .

F. REACTIVITY 1. Cornproportionation In contrast to certain other flavoproteins ( I 48), most flavodoxins comproportionate readily in the absence of mediators (61,68). I n P. elsdenii flavodoxin, the rate of reaction (3) depends on pH and ionic strength, varying a t pH 8.5 from 100 M-’ min-I a t zero ionic strength to almost lo6 M-’ min-’ a t I = 0.24 (1.69). This marked dependence on ionic strength probably reflects the large net negative charge on the protein. The reverse, or disproportionation, rates are smaller for flavodoxins, since the equilibrium in reaction (3) favors the semiquinone (68). A rate constant of 5 X lo3 M-l min-1 has been determined for the disproportionation of A . vinelandii flavodoxin a t pH 11; a t pH 8.3 in 0.05 M pyrophosphate the constant for P . elsdenii flavodoxin is only 47 M-I min-’ (68). Not surprisingly, rates for the comparable reactions of free flavins are much greater. Disproportionation of neutral FMN radicals proceeds with a rate constant of the order of lo5 M-I sec-1 (160). To obtain valid redox 146. R. D. Draper and L. L. Ingraham, ABB 139, 265 (1970). 147. L. Tauscher, S. Ghisla, and P. Hemmerich, Helv. Chim. Acta 56, 630 (1973). 148. V. Massey, in “Flavim and Flavoproteins” (H. Kamin, ed.), p. 231. Univ. Park Press, Baltimore, Maryland, 1971. 149. S. G. Mayhew and V. Massey, BBA 319, 181 (1973). 150. S. P. Vaish and G. Tollin, Bioenergetics 2, 61 (1971).

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potentials for the flavodoxins, it is, of course, essential that the comproportionation reaction attain equilibrium during titrations, but that may be accomplished by addition of mediators if necessary. 2. Sodium Dithionite The individual flavodoxins vary in their reactivity with dithionite and occasionally display peculiar behavior upon titration with this reagent. In view of the wide usage of dithionite as a reducing agent for redox proteins, its reactions with the flavodoxins will be described in some detail. Flavodoxins from P. elsdenii and Clostridium MP react rapidly with equimolar dithionite at p H 7, and full reduction is achieved within a few minutes (IS).The kinetics of the reduction of P. elsdenii flavodoxin a t p H 8.5 have been investigated by stopped-flow spectrophotometry at ionic strengths where comproportionation does not complicate the results (149). The reduction of both oxidized and semiquinone forms of the protein is first order with respect to protein concentration, but the semiquinone is reduced much more rapidly than is the oxidized form. The reaction rates depend on the square root of dithionite concentration. From these observations, Mayhew and Massey (149) concluded that P. elsdenii flavodoxin is reduced by the dissociation product of dithionite, SOZ', and that full reduction of the oxidized protein involves two sequential one-electron transfers according to the following scheme: K1 szo2- s 2SOZ'

Oxidized flavodoxin Flavodoxin semiquinone

+ SO2'

+ SO1'

+ SO2 f flavodoxin hydroquinone + SO1 k 2 flavodoxin semiquinone k

(6)

(7) (8)

The ratio of the rate constants, k z / k l , is approximately 450; consequently, it is not possible to detect the semiquinone during reduction of the oxidized protein by an excess of dithionite. The values of kl and kz depend use of the value given by Lynn et al. (161) leads on determination of K 1 ; to the estimate that kz is about 4 x lo7 M-l sec-1. I n a similar study of the dithionite reduction of additional redox proteins and small molecules, Lambeth and Palmer (166)found that one-electron reduction by SOZ' is the dominant pathway in several other reactions. Although ffavodoxin from P. elsdenii is fully reduced by one mole of dithionite per mole of flavin a t pH 7 and above, this stoichiometry does not obtain at lower pH; for example, a t pH 5.2,where dithionite is still reasonably stable under anaerobic conditions, about 12% of the flavin 151. S. Lynn, R. G. Rinker, and W. H. Corcoran, J. Phys. Chem. 88,2363 (1964). 152. D. 0. Lambeth and G . Palmer, JBC 248, 6095 (1973).

104

STEPHEN Q. MAYHJGW AND MARTHA L. LUDWIG

remains as the semiquinone in the presence of a twelvefold excess of the reductant (149).Further studies of the effect of pH on the reduction of this protein by dithionite strongly suggest that a pH-dependent equilibrium, described in part by Eq. (8) and subsequent hydration of SO,, is established (153).The ability of dithionite to achieve full reduction of the protein a t high pH is explicable if the potential of the dithionite redbx system remains pH-dependent and continues to decrease in the region where E , for flavodoxin becomes pH-independent. At pH 5, the redox potential of the dithionite acceptor-donor is probably similar to El for P . elsdenii flavodoxin (-323 mV) ; at pH 8, where El is -375 mV and independent of pH, the potential of the dithionite system is evidently more reducing. At pH 7 and p H 8.3, flavodoxin from C. p a s t e ~ T ~ u nbehaves u~ somewhat like P. elsdenii flavodoxin a t p H 5.2. Addition of one equivalent of dithionite generates the semiquinone as expected, but further additions do not produce stoichiometric yields of reduced protein. Some side reactions which consume dithionite may intervene (13). Flavodoxins from A . nidulans, A . vinelandii, and R. rubrum are much less reactive with dithionite than are the clostridial or P . elsdenii flavodoxins (19-22,39,51,125) ; methyl viologen can be used to mediate the reduction (100). Reduction of R . rubrum and A . vinelandii flavodoxin is incomplete a t pH 7 even in the presence of large excesses of the reagent (14,102). Full reduction of A . vinelandii flavodoxin can be achieved a t pH 8 with an excess of dithionite or alternatively a t pH 7 if the ionic strength is raised [e.g., in 3 M ammonium sulfate ( I O S ) ] . Tollin and co-workers (100,lOS) have proposed that the neutral flavin hydroquinone cannot be accommodated by the protein and that the extent of reduction is therefore controlled by the ionization of the reduced flavin (pK = 7.0). They attributed the effect of ionic strength to a shift in this pK. An alternative explanation would invoke the pH dependence of the dithionite system as described above. The higher pH values and dithionite concentrations required for full reduction of the A . vinelandii protein would then be a result of its very low E l ;the ionic strength effect would have to be ascribed to a salt-dependent increase in this potential. 153. After P . ekdenii flavodoxin is fully reduced by ti stoichiometric amount of dithionite at p H 8, anaerobic addition of acid to pH 5 converts half of the protein to the semiquinone, with a concomitant increase in absorption at 329

nm, an isobestic point for the protein, but a wavelength where dithionite absorbs. These changes can be reversed by raising the pH. The extent of reduction of the protein is influenced by the concentration of dithionite or bisulfite but not by sulfate or thiosulfate (164) 154. E. J. F. van Arem and S. G. Mayhew, unpublished.

2.

FLAVODOXINS AND ELECTRON-TRANSFERRING FLAVOPROTEINS

106

3. Oxygen and Ferricyanide

Oxidation of reduced flavodoxins by molecular oxygen is a nonphysiological reaction; in contrast to the flavoprotein oxidases and hydroxylases, the flavin-containing dehydrogenases presumably do not reduce oxygen during in vivo catalysis. The oxidation of dehydrogenases (and flavodoxin) by oxygen in vitro differs in a characteristic manner from the oxygen reactions of flavin-containing oxidases and hydroxylases (63). Flavodoxin is an interesting case since the reactivity of the fully reduced protein with oxygen is similar to that of model flavins, whereas the reactivity of the semiquinone is orders of magnitude less than that observed for comparable models. Kinetic studies have shown the following reactions to be significant : in the reoxidation of reduced flavodoxin (165,156)

The reactions of model flavins with oxygen proceed via intermediate flavin-02 adducts (167-159). Whether the reactions of flavodoxin with 0 2 involve similar adducts is not known. Determination of the kinetic parameters is complicated by reaction (3) and b y disproportionation of OaT to yield oxygen and peroxide. Furthermore, oxidation of both the anionic and neutral species of the fully and partially reduced flavins must be considered. [Equations (9)-(12) show only the forms predominating at neutral pH.] Stopped-flow spectrophotometry with P. elsdenii flavodoxin (166)has shown that the semiquinone is formed directly b y oneelectron oxidation of the hydroquinone since the rate of semiquinone formation is too great to be attributed t o full oxidation followed by comproportionation. The contribution of reaction (10) has been assessed by addition of superoxide dismutase to catalyze breakdown of 0 2 ’ . I n the presence of this enzyme, the rate of formation of the semiquinone a t pH 7.2 is decreased (Fig. 11). Decay of the semiquinone absorbance in 155. D . P. Ballou, Ph.D. Thesis, University of Michigan, Ann Arbor, 1971. 156. D. P. Ballou and S. G. Mayhew, unpublished. 157. Q . H. Gibson and J. W. Hastings, BJ 68,368 (1962). 158. V. Massey, G. Palmer, and D. P. Ballou in “Oxidases and Related Redox Systems’’ (T. E. King, H. S. Mason, and M. Morrison, eds.), Vol. 1, p. 25. Univ. Park Press, Baltimore, Maryland, 1973. 169. V. Massey, G. Palmer, and D. P. Ballou, in Flavins and Flavoproteins” (H. Kamin, ed.), p. 349. Univ. Park Press, Baltimore, Maryland, 1971.

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STEPHEN G . MAYHEW AND MARTHA L. LUDWIG

"

0

1

2 3 Time lsecl

L

FIG.11. Effect of superoxide dismutase on the reaction of reduced flavodoxin with oxygen. P . elsdenii flavodoxin (3.5x lo-' M in 0.05 M K phosphate buffer, pH 7.5) was titrated to full reduction with sodium dithionite (13) and then mixed in a stopped-flow spectrophotometer (149) with oxygen-containing buffer (0.15 M K phosphate, pH 7.2, and 6.26 x lo-' M 0 2 , curves 1 and 2; 0.15 M Na pyrophosphate, p H 9, and 2.5 x M 02,curves 3 and 4). Bovine erythrocyte superoxide dismutase was present in the experiments of curves 1 and 3.

the absence of dismutase (Fig. 11) provides evidence for reaction of 02' with the semiquinone [reaction (12)]. Reaction of the products of reaction (9), in a second one-electron step, is an interesting aspect of the oxidation scheme and may indicate that O 2 and 02' react at different sites. A minor pathway involving two-electron transfer has not been rigorously excluded. The rate constant for oxidation of P . elsdenii flavodoxin hydroquinone to the semiquinone at room temperature, in the presence of superoxide dismutase, is 3.5 x l o 4 M-1 sec-l and does not vary significantly with pH between 7 . 2 and 9.0 (155,156). The lack of pH dependence is consistent with a pK of 5.8 for formation of the anionic form of fully reduced P. elsdenii flavodoxin (58) (Section 11,E). For reoxidation of tetraacetylriboflavin a t pH 8.4 in the presence of dismutase, the rate constant is estimated to be ca. 1 X 104 M-I sec-l (158,169).For some other flavoprotein dehydrogenases, the comparable rates are of the order of lo2 RII-' sec-I (63).Because of the difficulty of determining the concentration of 02' in the reaction mixtures (168-160), constants for reaction (10) can160. V. Massey, s. Strickland, s. G. Mayhew, L. G. Howell, P. C. Engel, R. G. Matthews, M. Schuman, and P. A. Sullivan, BBRC 36, 891 (1909).

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not readily be evaluated, but it is evident from Fig. 11 that the secondorder rate constant for reaction (10) a t p H 7.2 must be larger than that for reaction (9). At p H 9, oxidation by 02' appears t o be slower than a t p H 7.2. For oxidation of the semiquinone, the rate constants vary considerably among the flavodoxins and in addition depend on oxygen concentration (58) and p H (58,102). At p H 7, k,,, for oxidation of the semiquinones of P. elsdenii or Clostridium MP flavodoxin is approximately 7 M-' min-', about 20 times greater than the value measured for A . vinelandii flavodoxin (102). [The contributions of reactions (11) and (12) have not been separated in these determinations.] All of the protein semiquinones are more rapidly oxidized a t higher pH, an effect attributed t o the presence of increasing concentrations of the more reactive semiquinone anion species (150).Edmondson and Tollin found a linear relationship between the reciprocal of the rate and the hydrogen ion concentration for oxidation of A . vinelandii flavodoxin radical (102).Assigning a p K of 11.5 for the ionization of the bound FMN radical, they calculated a rate constant of 2.4 X lo3 M-' sec-' for oxidation of the anion. Corresponding plots for P. elsdenii flavodoxin are not linear, and there are other indications that the pH effects with this protein are more complicated (155). A significant fraction of this semiquinone is oxidized via reaction (12), and the relative rates of dismutation of 02' and of its reaction with the protein may be very pH-dependent. Despite such individual variations, the flavodoxin semiquinones are all far less reactive with oxygen than might be predicted from models. The rate constants for reaction of oxygen with FMN or FAD radicals, generated by flash photolysis (150,161), are los M-' sec- for the anion and 104 M-1 sec-' for the neutral species ( 1 6 1 ~ ) . The factors which control the reactivity of fully reduced flavoproteins with oxygen are not completely understood. Hemmerich (16'2) has proposed that reactivity reflects hydrogen bonding a t N-1 or N-5 of the flavin and that the more planar the reduced flavin, the more readily will it be oxidized. Detailed structure analyses of fully reduced flavodoxins may suggest additional explanations for the effect of the protein on flavin reactivity. Oxidation of flavodoxins by ferricyanide probably provides a more appropriate model for the in vivo electron transfer reactions than does oxidation by oxygen. The ferricyanide and oxygen reactions respond differently to substitutions in the flavin ring (150,16'1),and the two reagents may react a t different sites in the isoalloxazine system. The oxidation of reduced flavodoxin by ferricyanide has been reported to be "very 161. J. M. Gillnrd and G. Tollin, BBRC 58, 328 (1974). 161a. R. A. Lazaarini and A. San Pietro, BBA 62, 417 (1962).

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STEPHEN G . MAYHEW AND MARTHA L. LUDWIG

fast” (63); rate constants for oxidation of flavodoxin semiquinone by ferricyanide are of the order of 104 M-1 sec-1 (63).Free neutral lumiflavin radical is oxidized much more readily with a constant of 5 X lo8 M-l sec-1 (161). Gillard and Tollin (161) have found the rate of transfer from lumiflavin radical to ferricyanide (and to oxygen) to be decreased, by a factor of 10 or less, in the presence of tryptophan concentrations sufficient for complex formation. They suggested that the tryptophan-FMN interaction in flavodoxins may be partly responsible for the lower reactivity of protein-bound FMN. Conformational changes accompanying the reaction could make a major contribution to the activation energy for oxidation of the semiquinone (Section II,C,5). 4. Redox Proteins

No complete kinetic studies of electron transfer between flavodoxin and individual acceptor proteins have been reported. While it has been assumed from the values of the redox potentials (Table IV) that flavodoxins function in vivo as one-electron carriers, alternating between the semiquinone and hydroquinone states, definitive evidence for this mechanism is not easy to obtain. Flavodoxins could in principle mediate transfer from either one- or two-electron donors to one- or two-electron acceptors over a wide range of potentials. Of the known flavodoxin-dependent reactions, the best characterized are reduction of ferredoxin-NADP+-reductase (FNR) , reduction of nitrogenase, and transfer of electrons between FNR and cytochrome c. Reduction of mammalian cytochrome c by reduced F N R does not proceed unless flavodoxin or ferredoxin is present (48,161~).Both mediators are known to form molecular complexes with F N R (48-46), and electron transfer between ferredoxin and FNR has been demonstrated (163). Under aerobic conditions the reduction of cytochrome c catalyzed by flavodoxin is partially inhibited by superoxide dismutase (160).Thus cytochrome c is reduced by 02’ in the presence of oxygen, but a direct reaction between flavodoxin and cytochrome c must also occur. For the overall reaction, NADPH + cytochrome c, turnover numbers have been determined for F N R but not for the mediators (164). Hence, the efficiency of flavodoxin in catalysis cannot yet be compared with the transfer rates in reactions involving dithionite, oxygen, or ferricyanide. 162. P. Hemmerich, A. P. Bhaduri, G. Blankenhorn, M. Briistlein, W. Haas, and W.-R. Knappe, in “Oxidases and Related Redox Systems” (T.E. King, H. S. Mason, and M. Morrison, eds.) p. 3. Univ. Park Press, Baltimore, Maryland, 1973. 163. J. Siedow, unpublished. 164. G. Forti and E. Sturani, Eur. J. Biochem. 3, 461 (1968).

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An EPR signal attributable to flavodoxin semiquinone can be detected in fully deuterated cells of S. Zividus. The concentration of radical increases in the dark and diminishes when electron flow is initiated by illumination (165). These observations suggest that the principal oxidation states of flavodoxin in photosynthesizing systems are the fully reduced and semiquinone forms. However, they do not demonstrate unequivocally that flavodoxin acts as one-electron carrier in the photosynthetic chain. Yates (36) has studied the reaction of flavodoxin from A . chroococcum with purified nitrogenase. Substrate amounts of reduced flavodoxin are converted to the semiquinone by catalytic quantities of nitrogenase in the presence of nitrogen and an ATP-generating system. Reduction of nitrogenase substrates requires fully reduced flavodoxin and will not proceed if only the semiquinone form is added. As in the photosynthetic reactions, these results appear to establish the kinetic importance of the semiquinone. Formation of the flavodoxin radical seems too rapid to be ascribed to two-electron oxidation and subsequent comproportionation, but additional experiments may be necessary to exclude the two-electron pathway. Determination of the precise mechanism of catalytic electron transfer by flavodoxins remains a challenge. The structure analyses suggest some restrictions on allowed mechanisms. Since the disposition of side chains adjacent to the isoalloxazine ring is so different in D. vulgaris and Clostridium MP flavodoxins, it is difficult to envision a universal pathway of electron transfer which utilizes these residues (Section II,B,3). The simplest suggestion offered by the structures is that the redox reactions involve the dimethylbenzene moiety of the flavin, which appears readily accessible for direct electron transfer. With the wealth of data on the structure and properties of the flavodoxins, the time seems ripe for incisive investigations of the oxidation-reduction reactions. 111. Electron-Transferring Flavoprotein

A. INTRODUCTION In the second edition of “The Enzymes” Beinert (1) described the properties of electron-transferring flavoprotein (ETF), an enzyme from mammals and mycobacteria which mediates the transfer of electrons from acyl-CoA and sarcosine dehydrogenases to the mitochondria1 cytochrome chain and also to nonphysiological electron acceptors. The mammalian enzyme is not readily obtained in high purity (1) and perhaps as a conse165. J.

R. Norris, H. L. Crespi, and J . J. Kata, BBRC 49, 139 (1972).

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STEPHEN G. MAYHEW AND MARTHA L. LUDWIG

quence only a few further studies on its molecular properties have been reported (166-168). This section is therefore concerned mainly with a bacterial flavoprotein which has recently been isolated from P. eZsdenii (169,170) and whose function resembles that of mammalian ETF. Peptostreptococcus elsdenii excretes short-chain fatty acids (C, to C,) into the growth medium during the fermentation of lactate and carbohydrates (54,171). The final reductive step in the synthesis of butyrate, the conversion of crotonyl-CoA to butyryl-CoA, is catalyzed by a flavoprotein (172,173) that is closely similar to butyryl-CoA dehydrogenase (BCD) of mammals. Electrons for this reduction can be provided by lactate and two further flavoproteins. One is a D-lactate dehydrogenase (pyridine nucleotide independent) in which FAD is the only known chromophore (174,175); the other is an enzyme which mediates electron transfer from D-lactate dehydrogenase to BCD (174,175). Although this mediator enzyme is concerned in the synthesis of fatty acids rather than their oxidation, it is functionally similar to ETF of mammals, and accordingly the term “electron-transferring flavoprotein” has been extended to include it (170,175) . D-

-

NADH

Lactate Lactate dehydrogenase D-

t

ETF-BCD

Crotonyl- CoA

I

(13)

BUtyWl-CoA

Peptostreptococcus elsdenii ETF also oxidizes NADH which therefore serves as an alternative source of electrons for the reduction of BCD and nonphysiological acceptors such as 2,6-dichlorophenolindopheno1 (170,17$,175). A similar diaphorase activity is associated with preparations of ETF from mammalian sources (166,176), but it has been attributed to contamination by other enzymes (1,177). 166. 167. 168. 169. 170. 171. 172. 173. 174.

D. D. Hoskins and R. A. Bjur, JBC 240, 2201 (1965). D. D. Hoskins, JBC 241, 4472 (1966). C. L. Hall, Fed. Proc., Fed. Amer. SOC.Ezp. Biol, 32,596 (1973). C. D. Whitfield, S. G. Mayhew, and V. Massey, Fed. Proc., Fed. Amer. SOC.

Ezp. Biol. 31, 447 (1972).

C. D. Whitfield and S. G. Mayhew, JBC 249, 2801 (1974). S. R. Elsden and D. Lewis, BJ 55, 183 (1953). R. L. Baldwin and L. P. Milligen, BBA 92, 421 (1964). P. Engel and V. Massey, BJ 125, 879 (1971). H. L. Brockman and W. A. Wood, Fed. Proc., Fed. Amer. SOC. Exp. Biol. 29,

862 (1970). 175. H. L. Brockman, Ph.D. Thesis, Michigan State University, E. Lansing, 1971. 176. F. L. Crane and H. Beinert, JBC 218, 717 (1956). 177. W. R. Frisell, J. R. Cronin, and C. G. Mackenrie, in “Flavins and Flavoproteins” (E. C. Slater, ed.), p. 367. Elsevier, Amsterdam, 1966.

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B. MOLECULAR PROPERTIES 1. Purification, Molecular Weight, and Amino Acid Composition Highly purified preparations of ETF are obtained from extracts of P. elsdenii by a combination of ion-exchange chromatography on DEAEcellulose, salt fractionation with ammonium sulfate, and gel filtration in Sephadex G-100 (170).I n some preparations, ETF is associated with a larger unidentified flavoprotein and is separated from this protein only during the final purification step. Purified ETF is stable for long periods in frozen solution at -200. The molecular weight of the enzyme, determined by gel filtration in calibrated columns of Sephadex, is about 73,500. The protein is composed of two nonidentical subunits which can be separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. From their electrophoretic mobilities and a comparison with standard proteins, the molecular weights of the two subunits were estimated to be 41,200 and 33,200. Amino acid and flavin analyses gave a minimum molecular weight of 37,800, a value which is in close agreement with the average molecular weight of the two subunits. The subunits evidently form a tight complex since strong denaturing agents such as sodium dodecyl sulfate or guanidine HC1 are required for their dissociation (170). Early estimates of the molecular weight of mammalian ETF ranged from 30,000 to 70,000 (176). A more recent determination with protein from pig liver gave a molecular weight of 35,000 (168). The amino acid composition of the bacterial protein has been determined and found to be unusually low in tryptophan (170). 2. Properties of the Flavin Chromophore

Purified preparations of P. elsdenii ETF contain about 1.4 moles of flavin per mole of protein, but they bind additional FAD to give a total of 2 moles per mole of protein (170); evidently some flavin is lost from the protein during its isolation. It is not known whether the two flavins are bound to the same or different subunits. Although FAD is the major chromophore in most preparations of the enzyme, all preparations contain at least traces of two modified flavins ( 170,178). When a protein-free extract of the enzyme is fractionated on a column of DEAE-cellulose, three colored bands are separated. In order of their elution from the column, the bands are yellow (FAD), green, and orange. After further purification, the chromophores in the green and orange bands were characterized and identified by comparison 178. C. D. Whitfield and S. C. Mayhew, JBC 249, 2811 (1974).

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STEPHEN G. MAYHEW AND MARTHA L. LUDWIG

TABLE V SPECTROSCOPIC PROPERTIES OF 6- AND 8-OH FLAVINS ~~

Compound

~~

.A.,

pH

(4

nm (mM-l cm-l)

PK

422(19,6) 7.1 323(19.6) 427(22.6) 600(3.41) 420(21.2) 6-OH-FMN 5.5 7.1 320(23) 423(24.9) 600(3.45) 9.0 asO(60.5) 446(26.9) SOH-FAD 3.1 4.8 6.7 252(80) 263sh(58.5)' 300(15.1) 478(36.7) 262(31) 435(26) SOH-FMN 3.0 4.8 7.0 252(51) 267sh(25.5)" 300(10.7) 472(41) 6-0H-FAD

5.6 9.0

262(50.5) 260(51.7)

Here, sh refers to a shoulder.

with chemically synthesized model flavins (178-183). The green chromophore is 6-hydroxy-7,8-dimethyl-10(ribityl-5'-ADP) 4soalloxazine (181,183,184) and the orange chromophore is 7-methyl-S-hydroxy-10(ribityl-5'-ADP) -isoalloxazine (179,I8O,185,186), The 6-hydroxyl and 8-hydroxyl groups are in direct conjugation with the chromophoric system and cause drastic changes in the spectrum of FAD; moreover, each of them gives rise to an additional pK in the flavin (Table V) . The spectra of both compounds are notable for their very intense peaks in the 400-480-nm region and, in the case of the anion of 6-OH-FAD, for a broad band of absorption centered at 600 nm. The proportions of the hydroxyflavins in ETF vary in different preparations of the enzyme (5-30 and 1-35% of the total flavin for 6-OH-FAD and 8-OH-FAD, respectively). At the highest levels observed, they together comprise about 50% of the total flavin (178). Such preparations differ in a number of properties from preparations which contain mainly FAD and only traces of the two modified flavins. First, and as detailed later, they have markedly different spectroscopic properties. Second, they 179. S. G. Mayhew and V. Massey, BBA 235, 303 (1971). S. Ghisla and S. G. Mayhew, JBC 248, 6568 (1973). 181. S. G. Mayhew, C. D. Whitfield, 9. Ghisla, and M. SchumanJorns, Eur. J . Biochem. 44, 579 (1974). 182. G. Schollnhammer and P. Hemmerich, Eur. J. Biochem. 44, 561 (1974). 183. G. Schollnhammer and P. Hemmerich, 2. Naturforsch. B 27, 1030 (1972). 184. A similar flavin but a t the level of FMN has been found in glycollate oxidase 180.

from pig liver (181),

185. N. A. Polyakova, L. S. Tul'chinskaya, L. G. Zapesochnaya, and V. M. Berekovskii, Zh. Obshch. Khim. 42, 465 (1972). 186. R. Addink and W. Berends, Tetrahedron 30, 75 (1974).

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fail to reduce BCD although they catalyze the oxidation of NADH with 2,6-dichlorophenolindophenol as electron acceptor. Third, they have a higher electrophoretic mobility and isoelectric point and can therefore be separated from ETF by electrophoresis or isoelectric focusing (178). Initially it was concluded that the modified flavins are associated not with ETF but with a different enzyme which also displays NADH dehydrogenase activity (179). However, more recent observations (178) have suggested that the protein moiety to which 6-OH-FAD and 8-OH-FAD are bound is very similar to apo-ETF, and that the different properties of the proteins separated by electrophoresis simply reflect differences in the proportions of the three flavin prosthetic groups. The molecular weight and subunit composition of the protein in the two bands was found to be the same; their amino acid compositions are very similar; protein with a high level of the hydroxyflavins shows a reaction of identity with antibody prepared against E T F ; and complexes made from apo-ETF and the separated hydroxyflavins catalyze the oxidation of NADH but they fail to reduce BCD (178,181). The mechanism of formation of the modified flavins is not known. However, i t appears that they are not present i n vivo in P. elsdenii since they are not detectable in crude extracts of the organism until after exposure of the extracts to oxygen (187), and it therefore seems likely that they are formed in ETF during the purification procedure. Attempts to generate them from FAD in the purified enzyme have failed. About 96% of the flavin of P . elsdenii ETF can be dissociated by chromotography of the holoprotein on a column of Sephadex G-25 equilibrated with guanidine HCI (170).After removal of the guanidine HC1, the apoprotein binds FAD with full restoration of catalytic activity (units per mole of bound flavin) ; it does not bind FMN. When freshly prepared, the apoprotein binds 2 moles of FAD per mole of protein ( K , = 5 X lo6 M-l, pH 6.2 and 16O). However, it is unstable and the amount of flavin which can be bound decreases progressively during storage a t 4 O or -2OO. The apoprotein also binds the hydroxyflavins ( K , = 6.7 X los M-' for 6-OH-FAD) and causes marked changes in their visible and near UV spectra (178,181).The spectra of the complexes of apo-ETF with the three separate flavins were used to determine the flavin composition of native ETF. Unlike normal flavins, the chromophores of 6-OH-FAD and 8-OH-FAD contain groups which ionize in the physiological pH range, and whose pK values can be studied after binding of the flavins to apoflavoproteins. When 6-OH-FAD is bound by apo187. C. D. Whitfield, private communication; I. Dekker and S. G . Mayhew, unpublished observations.

114

STEPHEN G. MAYHEW AND MARTHA L. LUDWIG

WMLENGM. nm

FIG.12. Absorption spectra of ETF. Curve 1, oxidized enzyme as isolated; curve 2, isolated enzyme after reduction with dithionite and reoxidation with potassium ferricyanide ; curve 3, enzyme fully reduced with dithionite.

ETF, the pK at 7.1 of the free flavin is decreased, and, in addition, the pH titration curve for the complex extends over a range of pH that is wider than expected for the ionization of a single group. It has been proposed that these effects might result from a positively charged group (pK 5-7) in ETF that is close to N-l:C-2:0-2of the flavin (181). These and similar observations with other apoflavoproteins (179-181,188) indicate that the hydroxyflavins can be used as reporter groups for the flavin binding sites of flavoproteins to obtain information not readily available from studies with unmodified FAD and FMN. As will be evident from the previous discussion, the absorption spectrum of ETF depends on the mixture of flavins in a particular preparation. Most preparations have a low content of the modified flavins and display absorption maxima at 275, 375, 450, and 660 nm, with relative intensities of 5.9:0.81:1 :0.01,respectively (Fig. 12). The band a t 660 nm is the result of a small amount of 6-OH-FAD; preparations with a higher content of this flavin have more absorption a t 660 nm and addi188. A similarly charged group in the flavin binding site of glycollate oxidase, but with a higher pK, has been inferred from the effects of pH on 6-OH-FMN-apoglycollate oxidase (181). In contrast, a negatively charged group near the flavin of flavodoxin, as observed in X-ray crystallographic studies (64,661, would account for the increase of p K observed when 6-OH-FMN and %OHF M N are bound by apoflavodoxin (180,181).

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FLAVOPROTEINS

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tional peaks a t 350 and 430 nm (181).In contrast, the absorption spectrum of preparations of the enzyme with a high content of 8-OH-FAD has a relatively intense peak a t 475 nm, marked shoulders on both sides of this peak, and further peaks at 318, 356, and 375 nm (178-180) ; such preparations are orange colored rather than yellow. The spectrum of ETF with FAD as the predominant flavin is altered when more FAD is added t o saturate a11 of the flavin binding sites (approximately 0.6 mole of added FAD per mole of protein). A large increase of absorption occurs around 400 nm, and the separation of the peaks a t 450 and 375 nm becomes correspondingly less distinct (170).Holoprotein prepared from apo-ETF and FAD shows a similar high absorption at 400 nm, and the extinction coefficient a t 450 nm of the newly bound flavin is low (10,500 M-l cm-l) by comparison with that of the FAD present in the enzyme as it is isolated (12,500 M-l cm-l). Reduction of the isolated enzyme and subsequent reoxidation also leads to a change in the spectrum, and again the most obvious effect is a large increase in the absorption a t 400 nm. The explanation for these changes is not known. The flavin prosthetic group of mammalian ETF is also FAD (1,168,189).Crane and Beinert (176) reported that the pig liver enzyme contains 1 mole of FAD per 83,500 g protein, but a higher value (1 FAD per 35,000 g protein) has been observed more recently (168).The absorption spectrum of this ETF, and ETF from other mammalian sources, differs considerably from those described above for P . elsdenii ETF, particularly in the region 400-500 nm. Highly purified ETF from pig liver and beef heart has absorption maxima a t 270, 375, 437.5, and 460 nm, and there appears to be very little absorption a t wavelengths greater than about 520 nm ( 1 ) . Electron-transferring flavoprotein from ra t (177,189) and monkey (166,167) liver on the other hand has a high peak of absorption in the region of 410 nm and very poor resolution of the absorption bands expected for FAD. These mammalian enzymes have not yet been analyzed for hydroxyflavins, and in particular for 6-OH-FAD which has an intense absorption band between 400 and 440 nm. The flavin of P. elsdenii ETF shows considerable fluorescence, a property which is shared by mammalian ETF (1,168) but by very few other flavoproteins (164.The fluorescence intensity is roughly twice that of free FAD, and it depends on the treatments described earlier which alter the absorption spectrum. The protein of this ETF is also fluorescent, and the positions of the fluorescence excitation and emission maxima suggest that there are contributions from both tyrosine and tryptophan ( 17 0 ) . 189. W.

R. Frisell, J. R. Cronin, and C. G. Mackenzie, JBC 237,2975 (1962).

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STEPHEN G. MAYHEW AND MARTHA L. LUDWIG

3. Oxidation-Reduction Preparations of P. elsdenii ETF with FAD as the major chromophore are reduced by NADH, D-lactate and catalytic amounts of D-lactate dehydrogenase (175),and sodium dithionite (Fig, 12). They can also be reduced photochemically with EDTA (170). Enzyme reduced with NADH shows a band of long wavelength absorption which has been attributed to a complex between the reduced enzyme and NAD' (170). Titration of the enzyme with dithionite showed that complete reduction requires two electrons per molecule of flavin; hence, there are no colorless redox-active groups in the protein. A red intermediate which is generated during photochemical reduction appears to be the flavin semiquinone anion. Formation of the anion, which also occurs in mammalian ETF (I), is unexpected since ETF functions as a dehydrogenase, and flavoprotein dehydrogenases usually form the blue neutral semiquinone ( 6 3 ) . Preparations of ETF with a high content of the hydroxyflavins, and complexes of apo-ETF with purified 6-OH- and 8-OH-FAD, are similarly reduced by NADH, dithionite, or EDTA and light, but no stable semiquinone intermediates have been observed (178,179,181). The reduced enzyme is rapidly oxidized by air, anaerobic ferricyanide, or crotonyl-CoA and catalytic quantities of BCD (170,176). The rapid oxidation in air contrasts sharply with the slow rate of reaction observed with mammalian ETF (176).As noted earlier, the spectrum of the reoxidised enzyme differs under some circumstances from that of the starting material (Fig. 12). The spectral change is observed when enzyme which contains less than the full complement of flavin (1.4 moles of FAD per mole of protein) is reduced for times as long as those required for anaerobic titrations with reducing agents. The changes do not occur if the enzyme is fully reduced by a single addition of NADH or dithionite and immediately reoxidized, nor are they observed with enzyme that is saturated with flavin (which already has high absorption a t 400 nm). The spectral change is independent of the method used to reduce and oxidize the enzyme, and it is not accompanied by significant changes in catalytic activity (170). C. CATALYTIC PROPERTIES Electron-transferring flavoprotein from P . elsdenii mediates the oxidation of D-lactate dehydrogenase or NADH and the reduction of BCD. The intermediary role of ETF in these reactions has been established in coupled assays with catalytic amounts of the three enzymes and also by studying partial reactions with substrate levels of ETF or BCD as

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the final electron acceptor (170,1?'2,17.5). Detailed kinetic studies with the purified enzyme and physical studies on its interactions with BCD and D-lactate dehydrogenase have not yet been carried out. D-Lactate dehydrogenase and NADH are the only known physiological electron donors for ETF, but a variety of compounds, including 2,6-dichlorophenolindophenol, will serve as electron acceptors (176,175) . The NADH dehydrogenase activity does not result from contamination by another enzyme as seems probable for ETF from pig (I) and rat (177) liver. Several protein fractionation techniques were used to analyze the purified protein (electrophoresis in acrylamide gel or Sephadex G-100, isoelectric focusing, gel filtration in Sephadex G-100,and analytical ultracentrifugation), but they neither separated the NADH dehydrogenase activity from ETF nor gave any indication for inhomogeneity other than that resulting from the hydroxyflavins (Section 1II1B,2). The modified flavins influence the activities of the enzyme since, as noted earlier, their complexes with apo-ETF display NADH dehydrogenase activity but they do not reduce BCD. Under defined assay conditions with 2,6dichlorophenolindophenol as electron acceptor, the relative NADH dehydrogenase activities of FAD-apo-ETF, 6-OH-FAD-apo-ETF1 and fI-OH-FAD-apo-ETF, are 1 :2.9: 0.31, respectively. The observed NADH dehydrogenase activity of enzyme preparations with the highest content of the hydroxyflavins agrees well with the theoretical value calculated from the activities of the separate complexes and the flavin composition of the enzyme (178).Although such preparations lack ETF activity, about half of their flavin content is unmodified FAD. It appears that modified flavin at one site in the enzyme influences the enzymic activity of FAD a t the other site since addition of 1 equivalent of 8-OH-FAD to apo-ETF that is 50% saturated with FAD causes a complete loss of ETF activity; NADH dehydrogenase activity in such mixed complexes is as expected for the binding of both flavins (178).This inhibitory effect of the hydroxyflavins is not understood. It is not yet clear whether the two molecules of FAD in ETF are equivalent in the catalytic reactions of the enzyme. When FAD is added to the isolated enzyme to saturate the flavin binding sites, the content of bound flavin increases by 40%, but the ETF activity is doubled and the NADH dehydrogenase activity with 2,6-dichlorophenolindophenolis unchanged (170).These observations, and the accompanying changes in the absorption spectrum (Section 111,B12), suggest that there may be differences between the two flavin binding sites. However, when increments of FAD are added to apo-FTF, the two activities increase in parallel until the end point at 2 moles of flavin per mole of protein; the changes in the absorption spectrum are also linear and provide no evidence for

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STEPHEN G. MAYHEW AND MARTHA L. LUDWIG

differences in either the spectroscopic properties of the sites or their affinities for FAD (17'0). Beinert (1) has discussed the catalytic properties of mammalian ETF and experiments in which ETF from pig liver was shown to substitute for ETF prepared from other mammalian sources and mycobacteria. Similar exchange experiments with the protein from P. elsdenii have not yet been attempted.