[42] Flavoproteins (Overview)

[42 ]

FLAVOPROTE|NS

[42] F l a v o p r o t e i n s !

397

(Overview)

By THOMAS P. SINCER and DALE E. EDMONDSON

Classification of Flavoenzymes Flavoenzymes may be defined as electron-transferring enzymes that contain a bound flavin prosthetic group (FMN or FAD). They catalyze redox reactions in which the flavin moiety accepts one or two electrons from the reducing substrate and donates one or two electrons to the oxidized substrate of electron acceptor. Both reduction and reoxidation of the flavin by metabolites may be direct or indirect. Thus, in xanthine oxidase the flavin is reduced by the carbon substrate by way of the Mo component of the enzyme, whereas the reoxidation of succinate or NADH dehydrogenases by coenzyme Q~0 in the respiratory chain occurs by way of the Fe-S centers of these enzymes. Enzymes in which flavin is the only redox active group are usually classified as "simple flavoproteins," and those that contain additional electron carriers are classified as "complex flavoproteins." In terms of this definition,D-lactate-cytochrome c reductase 2 and D-a-hydroxy acid dehydrogenase, 3 which contain Zn as well as FAD, would be considered simple flavoproteins, since Zn serves as the substrate binding site but plays no role in oxidation-reduction. Exceptions to this definition are lipoyl dehydrogenase, 4 glutathione reductase, 5 and thioredoxin reductase, 6 all of which contain disulfide groups that participate in catalysis but are nevertheless usually considered to be simple flavoproteins. Another widely used classification of flavoproteins, according to the reactions catalyzed, distinguishes among oxidases, dehydrogneases, and oxygenases. Flavoprotein oxidases catalyze a two-electron reduction of 1 The original studies reported here were supported by Program Project HL-16251 from the National Institutes of Health and by Grant No. PCM 76-03367 from the National Science Foundation. 2 T. P. Singer and T. Cremona, this series, Vol. 9, p. 302. 3 T. Cremona and T. P. Singer, this series, Vol. 9, p. 327. 4 V. Massey, Q. H. Gibson, and C. Veeger, Biochem. J. 77, 341 (1960). V. Massey and C. H. Williams, Jr., J. Biol. Chem. 240, 4470 (1965). 6 G. Zanetti and C. H. Williams, Jr., J. Biol. Chem. 242, 5232 (1967).

398

FLAVOPROTE1NS

[42]

molecular oxygen to hydrogen peroxide, according to the equation: I

\

H - - C - - X H + Oz ~

I

~X

+ H2Oz

/

As shown by Massey et al., 7,s flavoprotein oxidases share the following distinguishing properties: (1) they form anionic (red) flavin semiquinones on reduction by one electron; (2) they readily form flavin-sulfite adducts; (3) they do not catalyze the one-electron reduction of oxygen to the superoxide anion (02-). 9 The molecular basis of these properties is not known at present. It may also be noted that some flavoproteins oxidases do not form a tight sulfite adduct. 13 Flavoprotein dehydrogenases do not reduce molecular oxygen at catalytically significant rates. Their physiological electron acceptors are cytochromes, nonheme iron proteins, quinones, oxidized pyridine nucleotides, or other flavoproteins. Their catalytic activities are often determined with artificial electron acceptors, such as ferricyanide, phenazine methosulfate, etc. This class of flavoproteins form neutral (blue) semiquinones on one-electron reduction 7 and do not form a tight flavinsulfite adduct, 7 and their hydroquinone forms reduce oxygen to the superoxide anion. 8 A subclass of flavoproteins dehydrogenases contains a redox active disulfide moiety in the holoenzyme, which accepts electrons from the reduced flavin and transfers them to external disulfide-containing substrates (for a recent review see Williams14). Flavoprotein oxygenases catalyze the incorporation of molecular oxygen into the substrate to form an oxygenated product. They may be subdivided into monooxygenases (which catalyze the insertion of one 7 V. Massey, F. MOiler, R. Feldberg, M. Schuman, P. A. Sullivan, L. G. Howell, S. G. Mayhew, R. G. Matthews, and G. P. Foust, J. Biol. Chem. 244, 3999 (1969). s V. Massey, S. Strickland, S. G. Mayhew, L. G. Howell, P. C. Engel, R. G. Matthews, M. Schuman, and P. A. Sullivan, Biochem. Biophys. Res. Comm,n. 36, 891 (1969). 9 Xanthine oxidase may seem to be an exception, since it can form superoxide as well as H202 .10 The current view, however, is that in its native form this enzyme is a dehydrogenase and that autooxidizability of the flavin emerges during purification as an artifact ("D to O conversion"). 11,12 10 j. S. Olson, D. P. Ballou, G. Palmer, and V. Massey, J. Biol. Chem. 249, 4350 (1974). 11 W. R. Waud and K. V. Rajogopalan, in "Flavins and Flavoproteins" (T. P. Singer, ed.), p. 566. Elsevier, Amsterdam, 1976. iz R. C. Bray, M. J. Barber, M. P. Coughlan, H. Dalton, E. M. Fielden, and D. J. Lowe, in "Flavins and Flavoproteins" (T. P. Singer, ed.), p. 553. Elsevier, Amsterdam, 1976. 13 R. J. DeSa, J. Biol. Chem. 247, 5527 (1972). 14 C. H. Williams, Jr., in "The Enzymes" (P. D. Boyer, ed.), 3rd ed., Vol. 13, p. 89. Academic Press, New York, 1976.

[42]

FLAVOPROTEINS

399

atom of molecular oxygen into the substrate) and dioxygenases, which incorporate two atoms of oxygen. With one exception (2-methyl-3-hydroxypyridine-5-carboxylate dioxygenase'~), all the known flavoproteins oxygenases fall into the first category. Two kinds of flavoprotein monooxygenases may be distinguished. Since only one of the two atoms of oxygen in the 02 molecule finds its way into the substrate in monooxygenase action, the other is reduced to water, using reducing equivalents derived from the substrate itself or from an external electron donor, which is usually reduced pyridine nucleotide. The former is called internal monooxygenase. The oxidative decarboxylation in the reaction shown below is an example: H

O

R - - C - - C O O H + 02 ~ R - - C - - X + CO2 + HzO

I

XH

The reaction catalyzed by external monooxygenases may be visualized as follows: RH + N A D ( P ) H + H + + O2--~ R - - O H + NAD(P) + + H20

Flavoprotein oxygenases have been intensively studied in recent years. A timely review has been published.'6

T h e Fitness of Flavins as Catalysts of Biological Oxidations Flavoproteins catalyze a vast array of oxido-reductions, ranging from the desaturation of carboxylic acids and thioesters to the hydroxylation of aromatic compounds, from the dehydrogenation of alcohols and of aldehydes to carboxylic acids to the oxidation of the pyridine ring. Several of the reactions catalyzed by flavoproteins have been reproduced in nonenzymic model systems with free flavins as catalysts. The riboflavin molecule thus appears to be a uniquely versatile chemical entity. The specificity and efficiency of any given flavoenzyme is primarily determined by the protein moiety, which greatly enhances the chemical reactivity of the flavin in some instances and blocks it completely in others, as in the case of dehydrogenases, in which the autoxidation of flavins is in most instances completely prevented. '~ L. G. Sparrow, P. P. K. Ho, T. K. S u n d a r a m , D. Zach, E. J. N y n s , and E. E. Snell, J. Biol. Chem. 244, 2590 (1969). ~ V. M a s s e y and P. H e m m e r i c h , In " T h e E n z y m e s " (P. D. Boyer, ed.), 3rd ed., Vol. 12, p. 191. A c a d e m i c Press, N e w York, 1975.

400

FLAVOPROTEINS

[42]

One might ask what are the factors responsible for this versatility of flavins. The first property to come to mind in this context is the potential of flavins ( E r a , 7 ~-- -219 mV for FAD 17 and -205 mV for FMN18), which is, on the one hand, close enough to that of pyridine nucleotides to act as a catalyst in transhydrogenations between pyridine nucleotide coenzymes and, on the other hand, spans the gap between the potentials of pyridine nucleotide and of a number of substrate pairs and the higher potential cytochromes and coenzyme Q. The redox potential of the flavin in flavoproteins covers a very wide span. Thus, the Em,7 value of the flavin in Peptostreptococcus elsdenii flavodoxin has been reported TM to be -370 mV, and that of D-amino acid oxidase is - 4 mV. 2° An important reason for the wide range of potentials that flavoenzymes exhibit is that the free radical form of flavins is rather stable and this stability is enhanced in many flavoproteins; this permits shuttling between the hydroquinone and radical, hydroquinone and quinone, or radical and quinone, each of which redox couples will have a different potential. In the examples cited, D-amino acid oxidase cycles between the hydroquinone and quinone forms, while flavodoxin cycles between the hydroquinone and radical states. The stability of the flavin radical is also responsible for the ability of flavoproteins to accept and donate either one electron or two. It has been often pointed out that this permits bridging the oxidation of twoelectron-donor carbon substrates to the reduction of obligate one-electron acceptors, such as iron-sulfur proteins or heine proteins. Another characteristic of flavins that is of great importance in biological oxidations is the chemical reactivity of the 4a and 5 positions of the isoalloxazine ring system, which permits the formation of covalent adducts with the substrate, 21'2z resulting in a lowering of the activation energy for the reaction. Another property of flavins that may contribute to their versatility is that they can exist either in planar configuration (in the oxidized or radical forms) or in the folded, butterfly configuration (in 17 W. M. Clark, "Oxidation-Reduction Potentials in Organic Systems." Williams & Wilkins, Baltimore, Maryland, 1960. 18 R. D. Draper and L. L. lngraham, Arch. Biochem. Biophys. 125, 802 (1968). 19 S. G. Mayhew, G. P. Foust, and V. Massey, J. Biol. Chem. 244,802 (1969). 2o M. Brunori, G. C. Rotilio, E. Antonini, B. Curti, U. Branzoli, and V. Massey, J. Biol. Chem. 246, 3140 (1971). 2J T. C. Bruice, in "Progress in Bioorganic Chemistry" (E. T. Kaiser and F. J. Kezdy, eds.), Vol. 4, p. 1. Wiley, New York, 1976. ~2 p. Hemmerich, in"Progress in the Chemistry of Organic Natural Products" (W. Herz, H. Griesbach, and G. W. Kirby, eds.), Vol. 33, p. 453. Springer-Verlag, Berlin and New York, 1976.

[42]

FLAVOPROTEINS

401

the hydroquinone form). 23 An example of how this predisposes flavins to their catalytic role is the nonenzymic oxidation of NADH and NADPH by free flavins, discovered over 25 years a g o . 24 It has been recently shown that oxidation-reduction between pyridine nucleotide and ravin occurs by a concerted two-electron transfer involving juxtaposition of the two aromatic ring systems, both of which must be in the planar configuration. ~5

Mechanisms of Flavoenzyme Catalysis Reductive

Phase

Knowledge of the catalytic mechanisms of flavoenzymes has increased considerably during the past decade. As of this writing, the reduction of the flavins has been more extensively studied than their reoxidation during the catalytic cycle. In the majority of flavoproteins the ravin is part of the substrate site; i.e., the substrate is bound in close proximity of the ravin and is thus able to transfer electrons directly to the ravin. As already noted, in some enzymes, like xanthine or aldehyde oxidases, Mo is the component first reduced by the substrate, and the reduced Mo then reduces the flavin, in turn. In flavoenzymes where substrate and ravin are juxtaposed at the catalytic site, two types of mechanisms have been recognized for the initial reduction of the ravin. In reactions involving the oxidation of C - - H bonds, a carbanion intermediate has been suggested from the studies of Walsh e t al. zG and Porter e t al. 27 This intermediate is thought to arise by proton abstraction by a basic group in the protein from the substrate. Subsequently, the carbanion is thought to form a covalent adduct with the ravin by way of N(5) and/or C(4a) (cf. above), which facilitates electron transfer from the carbanion to the flavin. 21'22'27 The other mechanism concerns redox reactions that occur nonenzymically between substrate and flavins at appreciable rates and are facilitated by the enzyme, e.g., the oxidation of reduced pyridine nucleotides. 23 p. Kierkegaard, R. Norrestam, P. Werner, 1. Cs6regh, M. von Glehn, R. Karlsson, M. Leyonmerck, O. R6nnquist, B. Stensland, O. Tillberg, and L. Torbj6rnsson, in "Flavins and Flavoproteins" (H. Kamin, ed.), p, 1. Univ. Park Press, Baltimore, Maryland, 1971. 24 T. P. Singer and E. B. Kearney, J. Biol. Chem. 183, 409 (1950). 2~ G. Blankenhorn, Ellr. J. Biochem. 67, 67 (1976). 26 C. T. Walsh, A. Schonbrunn, and R. H. Abeles, J. Biol. Chem. 246, 6855 (1971). 27 D. J. T. Porter, J. G. Voet, and H. J. Bright, Biochern. Biophys. Res. Commun. 49, 257 (1972).

402

FLAVOPROTE1NS

[42 ]

These are thought to occur by two-electron transfer 2s with a charge transfer complex as an intermediate, 29the formation of which is facilitated by the geometry of the ring system of the reactants, as already noted. Relatively little is known about the detailed mechanism of the reduction of the flavin in enzymes where a reduced metal, such as Mo, or a reduced flavoprotein, rather than a metabolite, serve as reductant. The existence of a chain of flavoproteins that pass electrons to each other has been well documented. Thus, the flavoproteins butyryl-CoA dehydrogenase, long-chain fatty acyl-CoA dehydrogenase, and sarcosine dehydrogenase are all reoxidized by the electron-transferring flavoprotein (ETF)? °'3' Reduced ETF is, in turn, reoxidized by the recently discovered iron-sulfur flavoprotein ETF dehydrogenase. 32"33 Oxidative Phase

The reoxidation of reduced flavins has been less extensively studied than the reductive phase of the catalytic cycle. Thus, few generalizations are possible. One generalization already pointed out is that oxidases react rapidly with molecular oxygen and reduce it to H202, whereas dehydrogenases react poorly (or not at all) with oyxgen and, when they reduce oxygen, the product is the superoxide anion. Although the reasons for this difference are not completely known, it is clear that at least two requirements must be met when a flavoprotein reduces 02 to superoxide, instead of H202. First, since this is a one-electron reduction, the semiquinone form of the flavoenzyme must be relatively stable. Second, the potential of the flavin for the one-electron reduction must be low enough to permit 02- formation, since the Era,7 value for the O2/O2v couple is -330 mV, 34 whereas that for OdH2Oz is much higher 35 (+ 270 mV). Enzymes that cannot reduce oxygen to superoxide, such as D-amino acid 2s For the purposes of this overview, no attempt will be made to distinguish between hydride transfer and two-electron transfer. 29 G. Blankenhorn, in "Flavins and Flavoproteins" (T. P. Singer, ed.), p. 261. Elsevier, Amsterdam, 1976. 3o H. Beinert, in "The Enzymes" (P. D. Boyer, H. Lardy, and K. Myrbfick, eds.), 2nd ed., Vol. 7, p. 447. Academic Press, New York, 1963. 3, W. R. Frisell, J. R. Cronin, and C. G. Mackenzie, in "'Flavins and Flavoproteins" (E. C. Slater, ed.), p. 367. Elsevier, Amsterdam, 1966. 32 F. J. Ruzicka and H. Beinert, Biochem. Biophys. Res. Commun. 66, 622 (1975). 23 T. Flatmark, F. J. Ruzicka, and H. Beinert, FEBS Lett. 63, 51 (1976). 34 p. M. Wood, FEBS Lett. 44, 22 (1974). 3~ p. George, in "Oxidases and Related Redox Systems" (T. E. King, H. S. Mason, and M. Morrison, eds.), Vol. 1, p. 15. Wiley, New York, 1965.

[42]

FLAVOPROTE|NS

403

oxidase and pyridoxamine p h o s p h a t e oxidase, are reported to h a v e a considerably m o r e positive potential than the value quoted for the 0 2 / 0 2 couple, and no evidence is available for the formation of a stable semiquinone during their catalytic action. 2°'36 On the other hand, e n z y m e s that do f o r m superoxide (e.g., flavodoxin 8"19) have a low redox potential and a stable free radical. Adducts of reduced flavin and oxygen have been d e m o n s t r a t e d in model s y s t e m s 37 and for m o n o o x y g e n a s e s 38 and have been isolated in the case of bacterial l u c i f e r a s e ? 9 There is substantial evidence that this type of adduct is a key intermediate in the oxidative phase of the catalytic cycle of several m o n o o x y g e n a s e s . 38 For a discussion of the role of this f l a v i n - o x y g e n adduct in the p r o p o s e d reaction m e c h a n i s m of m o n o o x y genases, the reader is referred to a recent paper. 3s

M e t h o d s of S t u d y The assay method of choice for steady-state kinetic studies of flav o e n z y m e s depends both on the type of e n z y m e (oxidase, dehydrogenase, or oxygenase) and the reaction catalyzed. A few generalizations are permissible, however. Oxidases m a y be conveniently assayed polarographically, although most of them react also with dyes, like phenazine methosulfate or W u r s t e r ' s blue, and the s p e c t r o p h o t o m e t r i c determination of dye reduction tends to be more sensitive and convenient. Internal m o n o o x y g e n a s e s are a s s a y e d polarographically, external m o n o o x y g e n ases spectrophotometrically or fluorometrically, following the disappearance of reduced pyridine nucleotide. Flavoprotein dehydrogenases are most reliably assayed spectrophotometrically, following the rate of reduction of a suitable dye. The latter must be carefully selected, h o w e v e r , so as to measure as nearly as possible the full activity of the e n z y m e , lest reoxidation of the e n z y m e by the dye b e c o m e s rate-limiting. 4° In cases where the substrate has intense a b s o r b a n c e or fluorescence in :~ D. B. McCormick, M. N. Kazarinoff, and H. Tsuge, in "Flavins and Flavoproteins'" (T. P. Singer, ed.), p. 708. Elsevier, Amsterdam, 1976. :37V. Massey, G. Palmer, and D. Ballou, in "Oxidases and Related Redox Systems" (T. E. King, H. S. Mason, and M. Morrison, eds.), p. 25. Univ. Park Press, Baltimore, 1973. :~"B. Entsch, D. P. Ballou, and V. Massey, J. Biol. Chem. 251, 2550 (1976). z3~j. W. Hastings, C. Balny, and P. Douzou, in "Flavins and Flavoproteins" (T. P. Singer, ed.), p. 53. Elsevier, Amsterdam, 1976. 40T. P. Singer, in "Methods of Biochemical Analysis" (D. Glick, ed.), Vol. 22, p. 123. Wiley, New York, 1974.

404

FLAVOPROTEINS

[42]

either the oxidized or reduced form, a convenient assay is provided by following the disappearance of the substrate or the formation of the product (e.g., xanthine oxidase, NAD(P)H-cytochrome reductases). With a few flavoproteins, none of the methods above provides a convenient assay. In such cases activity determination is based on a more complex multienzyme system in which the flavoprotein to be assayed is made rate-limiting (e.g., ETF dehydrogenase, ~2 flavodoxin41). Flavoenzymes are well suited for pre-steady-state kinetic studies, which are essential for the delineation of reaction mechanisms, since the flavin component has differing and intense absorbances in the three redox forms. Further, transient long wavelength intermediates have been observed in kinetic studies of many flavoenzymes. 42 Because of these properties, stopped-flow spectrophotometry has been extensively used in mechanistic studies of flavoenzymes. In addition, the fluorescence of oxidized free flavins is retained in a few flavoenzymes and is quenched on reduction by the substrate. In some other flavoproteins it is the hydroquinone form that fluoresces, and this fluorescence is quenched on oxidation. 43 Besides reduction, complex formation with competitive inhibitors may also quench the fluorescence of flavoproteins. 44 In such instances stopped-flow fluorescence measurements provide an alternative means of studying pre-steady-state kinetics. Since the flavin semiquinone is paramagnetic, except where spin coupling with another paramagnetic component in the enzyme occurs, the rate of formation and of decay of the semiquinone form of the flavoprotein may be monitored by the rapid-freeze EPR technique. 4~ This technique has been successfully used in studying the participation of flavin semiquinones in the catalytic action of several flavoenzymes. 46-49 41 S. G. Mayhew and V. Massey, J. Biol. Chem. 244, 794 (1969). 42 V. Massey and S. Ghisla, Ann. N. Y. Acad. Sci. 227, 446 (1974). 43 S. Ghisla, V. Massey, J. Lhoste, and S. G. Mayhew, Biochemistry 13, 589 (1974). 44 B. A. C. Ackrell, E. B. Kearney, and D. E. Edmondson, J. Biol. Chem. 250, 7114 (1975). 43 R. C. Bray, in "Rapid Mixing and Sampling Techniques in Biochemistry" (B, Chance, R. H. Eisenhardt, Q. H. Gibson, and K. K. Lonberg-Holm, eds.), p. 195. Academic Press, New York, 1974. 46 H. Beinert, B. A. C. Ackrell, E. B. Kearney, and T. P. Singer, Eur. J. Biochem. 54, 185 (1975). 47 j. C. Swann and R. C. Bray, Eur. J. Biochem. 26, 407 (1972). 48 D. Edmondson, D. Ballou, A. Van Heuvelen, G. Palmer, and V. Massey, J. Biol. Chem. 248, 6135 (1973). 49 C. Capeillere-Blandin, M. lwatsubo, F. Labeyrie, and R. C. Bray, in "Flavins and Flavoproteins" (T. P. Singer, ed.), p. 621, Elsevier, Amsterdam, 1976.

[42]

FLAVOPROTEINS

405

Membrane-Bound Flavoenzymes Extraction and Activity Determination

Although a number of flavoenzymes have markedly different properties in the soluble state than in the membrane-bound form, there does not seem to be any characteristic shared by all membrane-bound flavoproteins that sets them apart from soluble ones. In order to evaluate changes in the molecular or catalytic properties of flavoenzymes that may occur on removal from the membrane, one must be aware of two interrelated problems. First, the fact that these enzymes are tightly bound to their membranes requires devising special procedures in order to solubilize them. These may be methods intended to perturb the lipid environment in which the enzymes are embedded (e.g., treatment with detergents, with l-butanol, or with phospholipase A2), proteolytic digestion, or incubation with chaotropic agents. Some of these procedures may cause inactivation or modification of the enzyme during extraction. In order to ascertain whether the extraction or subsequent purification has damaged the enzyme and even to estimate the degree of extraction, it is clearly necessary to have a valid assay for the enzyme that functions equally well in soluble and membrane preparations. Devising such an assay constitutes the second problem. After extraction from the membrane and purification of a flavoprotein, it is often difficult or even impossible to use the natural electron acceptor (or donor) in the assay, in some cases because it has not been identified, in other cases because it is not readily available, and frequently because the purified enzyme in solution does not react with the natural oxidant (e.g., purified NADH, succinate, choline, and a-glycerophosphate dehydrogenases do not reduce 4° coenzyme Q10, and microsomal N A D P H cytochrome c reductase extracted with proteolytic enzymes does not reduce ~° cytochrome P-450). In such cases artificial electron acceptors (dyes, ferricyanide, water-soluble coenzyme Q analogs) must be used in the assay. An analogous problem exists where the natural electron donor is missing and reduced dyes must be substituted in the assay. Problems that immediately arise are the specificity of the flavoprotein for dyes and the accessibility of the dyes to the active site of the enzyme in the membrane. The artificial electron acceptor (or donor) selected must be able to measure as nearly as possible the full activity of the enzyme: otherwise the reaction of the enzyme with the dye, rather than with the substrate, will be measured. 50M. J. Coon, H. W. Strobel, and R. F. Boyer, Dr, g Metab. Disp. 1, 92 (1973).

406

FLAVOPROTE1NS

[42]

It is often difficult to make sure that a given dye assay measures the full activity of a flavoenzyme. An obvious test is to compare the reactivities of the enzyme with the natural and the artificial electron acceptor in a relatively intact membrane preparation. This is not always a sufficient criterion, however, since the natural electron acceptor itself may not be measuring the full activity. Thus, the rates of oxidation of NADH and of succinate via the respiratory chain in the inner membrane are considerably less than the turnover numbers of succinate and NADH dehydrogenases. 51'52 Sometimes it is possible to measure the rate of reduction of an endogenous component of the enzyme by the substrate directly: if the component participates in the catalytic cycle, the resulting rate provides an absolute value with which the rates obtained in dye assays may then be compared. Among flavoproteins derived from membranes the rates of reduction of iron-sulfur centers by the substrate have been measured by rapid freeze EPR techniques in the cases of succinate, 47 NADH, '~3 and ETF dehydrogenases?2 When this is not possible, a good indication that a given dye assay measures the full catalytic activity is that the activity at Vrnax is the same with a series of dyes. Thus, soluble succinate dehydrogenase gives the same activity with phenazine methosulfate, Wurster's blue, and ferricyanide ("low K i n " site). '~4 Comparison of the reactivities of flavoproteins in soluble and membrane-bound form is also difficult because some of the most useful artificial electron acceptors (e.g., ferricyanide, phenazine methosuifate), cannot traverse the membrane, and the enzyme may be located on the inner surface (or inside) of the membrane. Thus, NADH and succinate dehydrogenases are located on the inner surface of the inner membrane: becasue of this, their activities in intact mitochondria cannot be measured with these electron acceptors unless provisions are made to assure their penetration. 4o Despite these difficulties, comparison of the total activity of the enzyme in the membrane before extraction and in the extracted form is important in order to assure that the solubilization has neither created nor destroyed catalytic activity, i.e., has not modified the catalytic properties of the enzyme. We have discussed elsewhere ~5 how failure to control this had led to the isolation of the antimycin-insensitive NADHcytochrome c reductase and rotenone-insensitive NADH-coenzyme Q 51 T. P, Singer, E. B. Kearney, and W. C. Kenney, Adv. Enzymol. 37, 189 (1973). ~2 T. P. Singer and M. Gutman, Adv. Enzymol. 34, 79 (1971). 53 H. Beinert, G. Palmer, T. Cremona, and T. P. Singer, J. Biol. Chem. 240, 475 (1965). 54 B. A. C. Ackrell, E. B. Kearney, and T. P. Singer, this volume [47], ~s T. P. Singer, in "Biological Oxidations" (T. P. Singer, ed.), p. 339. Wiley, New York, 1968.

[42]

FLAVOPROTEINS

407

reductase activities from mitochondria, activities that cannot be found in intact inner membranes. Assays with artificial electron acceptors may not reveal subtle modifications of enzymes incurred on isolation. (In fact, there is no a priori reason to assume that physiological oxidants necessarily detect structural changes that do not affect the catalytic site.) Methods for detecting such subtle changes may come to light only many years after the isolation of the enzyme and are usually the result of the elaboratibn of an alternate purification procedure. For example, the finding56"57 that NADPH-cytochrome c reductase from liver microsomes solubilized with detergents reacts with cytochrome P-450, reconstituting hydroxylase activity, whereas preparations isolated by earlier procedures utilizing proteolysis to extract the enzyme are active with artificial electron acceptors (cytochrome c, ferricyanide) but not with cytochrome P-450, came many years after the first isolation of the e n z y m e ? 8 Similarly, the discovery 59 that succinate dehydrogenase must be extracted under rigidly anaerobic conditions in the presence of succinate in order to recombine with the inner membrane and restore succinoxidase activity followed years after the isolation of the enzyme. 6° The lesson to be learned here is not that assays involving artificial electron acceptors are not informative about the degree of intactness of a flavoprotein, 61 but that as many criteria as are available should be applied to ascertain whether modification of the enzyme has occurred on removal from the membrane environment. As new, more sophisticated criteria are discovered, one has to go back and apply them to existing preparations, for they may reveal damage undetected by previous methods. Artificial electron acceptors continue to be useful, even essential, in isolating this class of enzymes, and some of them are capable of detecting the same subtle changes as reconstitution tests. 6z

Comparison of Properties of Some Flavoenzymes in the Soluble and Membrane-Bound States Although a number of flavoenzymes have been extracted from biological membranes and extensively purified, systematic comparison of 58 j. L. Vermillion and M. J. Coon, in "Flavins and Flavoproteins" (T. P. Singer, ed.), p. 674. Elsevier, Amsterdam, 1976. 57 y . Yasukochi and B. S. S. Masters, J. Biol. Chem. 251, 5337 (1976). 58 B. L. Horecker, J. Biol. Chem. 183, 593 (1950). 5, D. E. Keilin and T. E. King, Proc. R. Soc. London Ser. B 152, 163 (1960). 80 T. P. Singer, E. B. Kearney, and N. Zastrow, Biochim. Biophys. Acta 17, 154 (1955). 81 T. E. King, Biochim. Biophys. Acta 47, 430 (1961). 62 A. D. Vinogradov, B. A. C. Ackrell, and T. P. Singer, Biochem. Biophys. Res. Comrnun. 67, 803 (1975).

408

FLAVOPROTE1NS

[42]

the properties of these e n z y m e s in the soluble and membrane-bound form appears to have been made only for succinate and N A D H dehydrogenases. Available data range from instances where an e n z y m e appears to be extractable without a known change in properties to those where the membrane-bound and soluble form differ in a great many respects. As discussed below, the changes occurring on removal from the membrane environment are at least in one case reversed on reincorporation into the membrane. Monoamine oxidase from outer membranes is an example of an enzyme that may be extracted from mitochondria by the procedure given elsewhere in this volume 63 with no loss of activity and no change in Km for either the substrate (benzylamine) or the oxidant (molecular oxygen). Moreover, the e n z y m e shows the same sensitivity to a whole series of inhibitors in the outer membrane and in purified preparations. Microsoma| N A D H - c y t o c h r o m e b5 reductase may also be extracted and isolated in homogeneous form apparently with no change in activity toward either the natural electron acceptor (cytochrome bs) or ferricyanide. 64 The e n z y m e solubilized by incubation with iysosomes 6~ or cobra venom (presumed to be a proteolytic procedure 66) has a molecular weight of 33,000, and the form extracted with detergents 64 (Triton X-100) is larger (43,000). The 10,000 dalton fragment present in the detergentsolubilized e n z y m e is thought to be involved in binding of the e n z y m e to the membrane but not in catalysis, since the two forms have identical activity in the ferricyanide assay. The detergent-extracted e n z y m e is only a third as active with c y t o c h r o m e b~ as with ferricyanide, however. This has been ascribed to aggregation, for the addition of 0.004% (w/v) deoxycholate doubles the reactivity with c y t o c h r o m e b~ without affecting the reduction of ferricyanide.64'67 Microsomal N A D P H - c y t o c h r o m e c (P-450) reductase has also been solubilized with proteolytic enzymes 6s and with detergents.57 Both forms show the same turnover number with c y t o c h r o m e c (an artificial electron acceptor) and contain 1 mol each of F M N and FAD but differ in molecular weight (71,000 and 78,000, respectively), and only the detergent-solubilized form can reduce c y t o c h r o m e P-450, with resultant reconstitution of hydroxylase activity. 56'57 Although it would appear from this that the 6~j. 1. Salach, Jr., this volume [49]. 64L. Spatz and P. Strittmatter, J. Biol..Chem. 248, 793 (1973). 6~p. Strittmatter, J. Biol. Chem. 246, 1017 (1971). ~6p. Strittmatter, J. Biol. Chem. 236, 2329 (1961). 6r M. J. Rogers and P. Strittmatter, J. Biol. Chem. 249, 895 (1974). n8C. H. Williams, Jr., and H. Kamin, J. Biol. Chem. 237, 587 (1962).

[42]

FLAVOPROTEINS

409

form extracted with detergents is closer to the enzyme as it occurs in the microsomes, comparison of its properties with the membrane-bound enzyme does not appear to have been reported. Choline dehydrogenase from liver mitochondria69 and a-glycerophosphate dehydrogenase from brain mitochondria7°'7' have also been isolated both by extraction with detergents and with cobra venom. (The venom in these cases is a source of phospholipase A2, since pure phospholipase A2 isoenzymes replace the crude venom. 7~) Choline dehydrogenase activity of mitochondria, measured with phenazine methosulfate (PMS) as an oxidant, is fully recovered on solubilization with snake venom, but only partially recovered on extraction with a detergent. 6a Conditions for measuring the reactivity with the natural electron acceptor (presumably coenzyme Qlo) have not been established yet. Extraction of o~-glycerophosphate dehydrogenase from pig brain acetone powder with phospholipase A2 is accompanied by partial loss of activity, TM as measured in the PMS assay, or its spectrophotometric modification. TM It has been reported 71 that extraction with Triton X-100 results in full recovery of the activity. These data were obtained with a polarographic modification of the phenazine assay, at fixed dye concentration, which measures an indeterminate fraction of the activity. 4° When assayed spectrophotometrically, at Vmax (PMS), loss of activity on extraction was comparable to that obtained by the venom procedure. 74 Coenzyme Q10 seems to be the natural electron acceptor. 73'75 It is reduced, albeit relatively slowly, by the detergent-extracted enzyme, 7' while the venom-extracted preparation has not been tested. Comparison of the reactivity of soluble preparations with those of the membranebound enzyme would probably have to be made with short chain, watersoluble coenzyme Q analogs. Further studies are required to decide whether the partial loss of activity toward artificial electron acceptors on removal from the membrane is indeed an inactivation, rather than a reflection of the effect of some membrane component on the activity, as in the case of succinate dehydrogenase.76'77 na T. Kimura and T. P. Singer, this series, Vol. 5, p. 562. 70 R. L. Ringlet and T. P. Singer, this series, Vol. 5, p. 432. 7, A. P. Dawson and C. J. R. Thorne, Biochem. J. 111, 27 (1969). 7z j. I. Salach, R. Seng, H. Tisdale, and T. P. Singer, J. Biol. Chem. 246, 340 (1971). 7.',j. I. Salach and A. J. Bednarz, Arch. Biochem. Biophys. 157, 133 (1973). 74 j. 1. Salach, unpublished data. 75 L. Szarkowska and A. K. Drabikowska, L(['e Sci. 7,519 (1963). 76 B. A. C. Ackrell, E. B. Kearney, P. Mowery, T. P. Singer, H. Beinert, A. D. Vinogradov, and G. A. White, in "Iron and Copper Proteins" (K. Yasunobu, H. F. Mower, and O. Hayaishi, eds.), p. 161. Plenum, New York, 1976. 77 B. A. C. Ackrell, E. B. Kearney, and T. P. Singer, J. Biol. Chem. 252, 1582 (1977).

410

FLAVOPROTE1NS

[42]

In comparing the properties of membrane-bound and soluble succinate dehydrogenase, the discussion will be restricted to the reconstitutively active form of soluble enzyme, ~4 since the differences in its behavior from the enzyme as it occurs in membranes are removed on reincorporation into membranes. 7sThis serves to distinghish changes due to removal of the enzyme from the membrane environment from secondary changes that occur on modification of the enzyme during or after extraction. Inner membrane preparations from heart (ETP, ETPH) will be used as reference material for the membrane-bound form, since, as far as can be judged, the characteristics of the enzyme in such preparations are the same as in heart mitochondria. Keilin-Hartree preparations 79 may also be used as reference material, except when comparing catalytic activities, since the enzyme in this preparation has a low turnover number, apparently because of the presence of inactive enzyme. 76.77 Complex II is not suitable as a reference material for the membrane-bound form, since, as shown below, the properties of the enzyme in these particles are not typical of either the membrane-bound or soluble enzyme. The major differences between the membrane-bound and soluble forms of the enzyme are as follows. (1) The enzyme is much more stable in the membrane, particularly to oxygen. (2) Thenoyltrifluoroacetone (TTF) and carboxamides, acting at the same site, inhibit electron transport between the dehydrogenase and coenzyme Q,o in membranes. 8°'8' This results in complete loss of succinoxidase and succinate-coenzyme Q reductase activities, while the reduction of PMS is about 50% inhibited at Vmax (PMS) (Fig. 1A). Soluble preparations are not inhibited by these agents. 81"82Succinate dehydrogenase activity in Complex II is competitively inhibited, so that at Vm~x (PMS) no inhibition is seen (Fig. 1B). (3) Incubation of inner membrane preparations with cyanide 83 results in an inactivation pattern, 84's5 which suggests reaction with a nonheme iron component 84 of the enzyme, probably the high-potential iron-sulfur (HiPIP) center, st Succinate oxidation is inhibited in the same manner and to the same extent in cyanide-treated preparations as in TTF-inhibited 78 T. E. King, this series, Vol. 10. p. 322. 7a T. E. King, this series, Vol. 10, p. 216. 80 T. P. Singer, this series, Vol. 55 [57]. 8, p. C. Mowery, D. J. Steenkamp, B. A. C. Ackrell, T. P. Singer, and G. A. White, Arch. Biochem. Biophys. 178, 495 (1977). 82 D. M. Ziegler, in -Biological Structure and Function" (T. W. Goodwin and O. Lindberg, eds.), Vol. 2, p. 253. Academic Press, New York, 1961. 83 C. L. Tsou, Biochem. J. 49, 512 (1951). s4 A. Guiditta and T. P. Singer, J. Biol. Chem, 234, 662 (1959). 85 E. Rossi, B. Norling, B. Persson, and L. Ernster, E,r. J. Biochem. 16, 508 (1970). 88 T. E. King, T. Ohnishi, D. B. Winter, and J. T. Wu, in "Iron and Copper Proteins" (K. T. Yasunobu, H. F. Mower, and O. Hayaishi, eds.), p. 182. Plenum, New York, 1976.

[42]

FLAVOPROTEINS

411

240 I

240

20.0 r

200

~160 ~ 12.0

/

160 12.0

80

80

/

40

o

i 2.0

i 40 PMS (raM-~)

6.0

o

20 40 60 PMS (mM-I )

FIG. 1. Comparison of effect of 3-methylcarboxin on ETP and Complex II. (A) O, untreated ETP (turnover number = 18,700); mE, ETP + 0.84 ~V/ 3-methylcarboxin. (B) O, Untreated Complex 1I (turnover number = 10,800); II, Complex II + 0.84 tzM 3-methylcarboxin. The ordinate represents reciprocal specific activity × 10 in (A) and reciprocal specific activity × 100 in (B).

samples. The catalytic activity of soluble preparations, 8~ including those possessing a viable HiPIP center, is not affected by cyanide, although cyanide reacts with their iron-sulfur components and bleaches the spectrum of the enzyme. 86 Inactivation of the succinate-PMS reaction in Complex II is competitive with the dye, disappearing at Vmax.81 (4) Extraction with pentane of coenzyme Q10 from membranes results in the same inhibition pattern for succinate dehydrogenase as incubation with cyanide, including -50% loss of succinate-PMS activity, rG's5The Km for succinate is also altered by this treatment. On reincorporating coenzyme Q10 into the membrane these effects are reversed, s5 Succinate oxidation in soluble and Complex II preparations is unaffected by pentane extraction. (5) The turnover number of succinate dehydrogenase, as assayed with either PMS or Wurster's blue, 54"87is -21,000 mol of succinate per minute per mole of histidyl flavin at 380. 77 This value declines to around 15,000 for the reconstitutively active form of the soluble enzyme, but returns to the original value on reincorporation of the enzyme into membranes. 7~'77 Extraction and reincorporation of the dehydrogenase is also accompanied by a completely reversible change in the Km for PMS. (6) The "low Km" reaction site of the enzyme for ferricyanide 54"62 is not detectable in inner membranes; it becomes fully reactive on extraction ~7 B. A. C. Ackrell. C. J. Coles, and T. P. Singer, FEBS Lett. 75, 249 (1977).

412

FLAVOPROTEINS

[42]

and again disappears on reincorporation of the enzyme into membranes. 88 (7) Reactivity with coenzyme Q homologs and analogs disappears on extraction of the enzyme and reappears on reincorporation into the membrane. This impressive list of differences cannot be ascribed to inactivation of the enzyme on extraction, since they are reversible. They may be rationalized as follows. The increased stability in the membrane is a property observed with many enzymes and may be assumed to be due to the shielding of labile groups by the hydrophobic environment. The same explanation, i.e., burying the reaction site in the membrane, which ferricyanide cannot penetrate, has been advanced to explain the absence of the "low Km" ferricyanide reaction site. 88 As noted above, the turnover number (TN) is -21,000 in membranes, -10,000-11,000 in cyanide-treated, TTF-inhibited, or coenzyme Q-depleted particles, and -15,000 in reconstitutively active, soluble preparations. It has been suggested that these three groups of turnover numbers may represent different conformations of the enzyme. 77 The highest activity, seen only with mitochondrial and relatively intact inner membrane preparations, has been thought to represent positive modulation by a membrane component, 7r'8~'89 possibly coenzyme Q.85 On depletion of the coenzyme Q by extraction or interruption of the communication between the dehydrogenase and the quinone by cyanide, TTF, or carboxamides, this modulatory influence would be removed and a second, low-activity form (TN = --10,000-1 1,000) may arise. The fact that the most carefully prepared soluble samples have a turnover number well in excess of this 54'77 suggests that the enzyme in solution may assume a third conformation, differing from those it has in the membrane environment. The fact that the succinate-PMS activity of a typical preparation of Complex II is unaffected by TTF, carboxamides, cyanide, or pentane extraction [at Vmax (PMS)] may reflect the virtual absence of coenzyme Q10 in these particles. 77"81Since Complex II samples are vesicular, it is not surprising that the turnover number of the dehydrogenase in typical samples of Complex II is about the same 77"a°as in coenzyme Q-depleted inner membrane. Whether the lower turnover number in Complex II than in mitochondria or inner membranes is due to this circumstance or to the 88 A. D. Vinogradov, E. V. Gavrikova, and V. G. Goloveshkina, Biochem. Biophys. Res. Commun. 67, 803 (1975). 8a B. A. C. Ackrell, E. B. Kearney, C. J. Coles, T. P. Singer, H. Beinert, Y. Wan, and K. Folkers, Arch. Biochem. Biophys, 182, 107 (1977). 9o W. G. Hanstein, K. A. Davis, M. A. Ghalambor, and Y. Hatefi, Biochernistry 10, 2517 (1971).

[42]

FLAVOPROTEINS

413

presence of inactive e n z y m e , as suggested by kinetic E P R data, 44's9 or both remains for future w o r k to decide. NADH Dehydrogenase of the Respiratory Chain N A D H dehydrogenase is an excellent e x a m p l e of the two interrelated problems involved in isolating m e m b r a n e - b o u n d flavoproteins, which have been discussed earlier in this chapter: devising a method of extraction from the m e m b r a n e that neither creates nor destroys catalytic activity and elaborating a reliable assay procedure, which measures the full activity before and after solubilization. In early studies of this e n z y m e artificial electron acceptors, such as 2,6-dichlorophenolindophenol or c y t o c h r o m e c, were used under arbitrary conditions, and isolation p r o c e d u r e s were devised that actually created these activities. 91-94 Reaction of the reduced F M N group of the e n z y m e with such oxidants is a potential shared with other flavoproteins; like the reaction with oxygen, it is not e x p r e s s e d in the native structure of the e n z y m e , but e m e r g e s only on extensive structural modification, 95'96 usually during the extraction step. The soluble flavoproteins isolated from animal tissues in this m a n n e r were thought by their discoverers to differ from each other, and no one knew for sure their relation to the N A D H d e h y d r o g e n a s e of the respiratory chain, because the two identifying marks of that e n z y m e - - r o t e n one sensitivity and energy conservation site 1 - - a r e lost on extraction. When these various N A D H oxidizing preparations were later c o m p a r e d under standardized conditions, 95,96 it b e c a m e clear that they did not differ materially from each other either in composition or in catalytic properties, although they were m a r k e d l y different from the m e m b r a n e - b o u n d N A D H d e h y d r o g e n a s e , from which they had been derived. This relatively low molecular weight ( - 7 8 , 0 0 0 daltons) preparation was originally called " D P N H - c y t o c h r o m e c r e d u c t a s e " ~1 and is referred to as " l o w molecular weight form of N A D H d e h y d r o g e n a s e " in more recent papers. ~-,~7 ~1H. R. Mahler, N. K. Sarkar, L. P. Vernon, and R. A. Alberty, J. Biol. Chem. 199, 585 ( 1952). ,2 B. DeBernard, Biochim. Biophys. Acta 23, 510 (1957). ~3B. Mackler, Biochim. Biophys. Acta 50, 141 (1961). ~4T. E. King and R. L. Howard, J. Biol. Chem. 237, 1686 (1%2). 95 H. Watari, E. B. Kearney, and T. P. Singer, J. Biol. Chem. 238, 4063 (1%3). "~"T. Cremona, E. B. Kearney, M. Villavicencio, and T. P. Singer, Biochem. Z. 338,407 (1963). 97 E. C. Slater, in "Flavins and Flavoproteins" (E. C. Slater, ed.), p. 387. Elsevier, Amsterdam, 1966.

414

FLAVOPROTEINS

[42]

Isolation of the enzyme in less modified form called for the simultaneous elaboration of a new assay procedure that fulfills the criteria given at the beginning of this section and of a milder extraction method that would preserve all the activity in terms of this assay, without creating activities not seen in submitochondrial particles. This was accomplished by Ringler et al. ,98.99 who extracted the enzyme quantitatively from beef heart ETP with phospholipase A2 at 30 ° and accounted for all the activity in a ferricyanide assay, without unmasking dye reductase or antimycininsensitive cytochrome c reductase activities. That this ferricyanide assay, under the conditions recommended, 4° indeed measures the full activity of the enzyme was proved later by the demonstration 53 that the rate of reduction of iron-sulfur center 1 by the substrate, measured by the rapid-freeze EPR technique, parallels catalytic activity in the ferricyanide assay with a series of NADH analogs and even during decay of the enzyme. The enzyme thus extracted retains most of the catalytic properties seen in ETP or Complex I, 100as well as all four EPR-detectable iron-sulfur centers, 101 including center 2, the probable site of coupling to the energy conservation system. 1°2 Although the properties of the purified enzyme have precluded accurate molecular weight measurements by conventional procedures, from FMN content it has been estimated to be at least 550,000 daitons. 52 It is therefore referred to in the literature s2'97as "high molecular weight form of NADH dehydrogenase." One may gain an insight into the effect of the membrane environment on NADH dehydrogenase by comparing Complex I, a purified but still particulate form of the enzyme, with the high-molecular-weight soluble preparation, since, as far as is presently known, the properties of the mitochondrial enzyme are retained during the isolation of this complex. The turnover number of the enzyme in the ferricyanide assay, per mole of FMN, is the same in the two preparations, 52'1°° as are the substrate specificity and the Km values for substrates, ~°3'1°4 Neither preparation catalyzes the reduction of dyes, antimycin-insensitive cytochrome c reduction, or rotenone-insensitive coenzyme Q reduction at appreciable rates. One notable difference is that while Complex I reacts well with 98 R. L. Ringler, S. Minakami, and T. P. Singer, J. Biol. Chem. 238, 801 (1963). 9~ S. Minakami, R. L. Ringler, and T. P. Singer J. Biol. Chem. 237, 569 (1962). 100 j. M. Machinist and T. P. Singer Proc. Natl. Acad. Sci. U.S.A. 53, 467 (1965). 101 M. Gutman, T. P. Singer, and H. Beinert, Bioehem. Biophys. Res. Commun. 44, 1572 (1971). 102 M. Gutman, T. P. Singer, and H. Beinert, Biochemistry 11,556 (1972). 1o3 S. Minakami, T. Cremona, R. L. Ringler, and T. P. Singer, J. Biol. Chem. 238, 1529 (1963). 104 A recent review 1o~state s that the K m of the purified enzyme for N A D H in the ferricyanide assay (10 -4 M) is much higher than in Complex I. It has been shown, 1°3 however, that the Km for N A D H is the same in the purified preparation as in the inner membrane. 1o5 y . Hatefi and D. L. Stiggall, in " T h e E n z y m e s " (P. D. Boyer, ed.), 3rd ed., Vol. 13, p. 175. Academic Press, New York, 1976.

[42]

FLAVOPROTEINS

415

added coenzyme Q1 (although not nearly as well as with ferricyanidel°6), the soluble enzyme does not. This has been taken by some authors as an indication that the enzyme is modified by the extraction procedure. One should consider, however, that Fleischer et al.l°7 and Machinist and Singer ~°° reported that lipids are required for the reduction of coenzyme Q homologs. Since the purified, high-molecular-weight enzyme is lipidfree, it would not be expected to reduce the quinone in a rotenonesensitive manner. Resolution of this question may have to await successful reconstitution of coenzyme Q reductase activity, perhaps by inserting the dehydrogenase into liposomes. To the authors' knowledge, such reconstitution has not been reproducibly achieved with any purified NADH dehydrogenase. The soluble dehydrogenase also does not bind rotenone or piericidin A. Again, it is known that the tight binding of these inhibitors in submitochondrial particles requires lipids.l°8 Other known differences are as follows. Certain - - S H groups are available to - - S H inhibitors in the soluble preparation but not in inner membranes. 109Further, while the nonheme iron : FMN ratios are the same in the purified, high molecular weight enzyme and in Complex I, the Fe:S ratio is close to 1 in the latter preparation but appears to be well above unity in the former. 11° The significance, if any, of this difference is not known. 111 It has also been stated w-' that the EPR signal of ironsulfur center 2 is modified in the soluble preparation. We have not detected such a change on comparing the soluble enzyme and phosphorylating submitochondrial particles. It may be seen that the major difference between the high molecular weight, soluble enzyme and its membrane-bound form is in rotenonesensitive coenzyme Q reductase activity. This has prompted a continuing search for alternative isolation methods in order to arrive at a soluble preparation possessing this activity. One such attempt was the application of the heat-acid-ethanol procedure, used earlier by others 9~-~z for the isolation of the low molecular weight form, to ETPH, a phosphorylating membrane preparation. 113 Since the resulting soluble flavoprotein re-

106 y . Hatefi, A. G. Haavik, and D. E. Griffiths, J. Biol. Chem. 237, 1676 (1962). lo7 S. Fleischer, A. Casu, and B. Fleischer, Fed. Proc., Fed. Am. Soc. Exp. Biol. 23, 486 (1964). 108 D. J. Horgan, H. Ohno, T. P. Singer, and J. E. Casida, J. Biol. Chem. 243, 5967 (1968). ~o.~M. Gutman, H. Mersmann, J. Luthy, and T. P. Singer, Biochemistry 9, 2678 (1970). 110 C. J. Lusty, J. M. Machinist, and T. P. Singer, J. Biol. Chem, 240, 1804 (1965). 111 While the high ratio of labile S to Fe may turn out to be an artifact, it is not due to the use of a low extinction coefficient for methylene blue, as has been suggested, 1°~ since the same extinction coefficient gave correct values for the labile S content of other preparations. 110 112 C. 1. Ragan, Biochirn. Biophys. Acta 456, 249 (1976). 113 R. L. Pharo, L. A. Sordahl, S. R. Vyas, and D. R. Sanadi, J. Biol. Chem. 241, 4771 (1%6).

416

FLAVOPROTEINS

[42]

duced coenzyme Q, it was regarded to be a more native form of NADH dehydrogenase than preparations previously described. Salach et a1.,114 however, showed that coenzyme Q reduction by this preparation was artifactual, because it was not inhibited by high concentration of rotenone or amytal. The preparation was, in fact, identical with Mahler's NADHcytochrome c reductase.'15 The transient coenzyme Q reductase activity of this low molecular weight enzyme could be shown to emerge on application of the heat-acid-ethanol procedure to the purified high molecular weight enzyme during its breakdown to the low molecular weight form. 114This experience illustrates one of the pitfalls in this field: the use of "physiological" assay systems may as easily lead to artifacts as of artificial ones. A detailed discussion of the many differences between the high and the low molecular weight forms of the enzyme is beyond the scope of this chapter and has been presented elsewhere? 2'96 It may suffice to point out that the differences in catalytic properties include the reaction with the substrate, as well as with electron acceptors, since the substrate specificity is altered in going from the high to the low molecular weight form, and that on dissociation of the high to the low molecular weight form most of the iron-sulfur content is lost; what remains is labile and not reduced by the substrate, and that the protein-FMN bond is also labilized in this transformation. It is clear, therefore, that the rest of the large NADH dehydrogenase molecule stabilizes the structure of the -80,000 molecular weight part, containing the flavin and substrate sites, and profoundly affects its reactivity. The membrane environment, in turn, stabilizes the structure of the high molecular weight form. Thus, while the enzyme in ETP is moderately stable to 37 °, the soluble form readily breaks down (or dissociates) to the low molecular weight form at this temperature. The structural basis of the relationship between the high and the low molecular weight forms has been a most difficult problem, one that has often led to animated discussions, perhaps because NADH dehydrogenase is so complex that it is difficult to unravel its structure by analogy with other proteins. According to one view, the low molecular weight form is a relatively unmodified enzyme or catalytically active subunit, which is merely dissociated from the respiratory chain by the treatments used in its isolation (e.g., heat, heat-acid-ethanol, urea, and other chaotropic agents). The weakness of this argument is apparent: the low molecular weight form is patently modified in most respects, as compared with the membrane-bound enzyme. 95"96'112 Moreover, although different preparations of the low molecular weight form are very close to each ,14 j. I. Salach, T. P. Singer, and P. Bader, d. Biol. Chem. 242, 4555 (1967). 115 D. R. Biggs, J. Hauber, and T. P. Singer, J. Biol. (_Twm. 242, 4563 (1%7).

[42 ]

v LAVOPROTEINS

417

other in catalytic properties, molecular weight, and prosthetic group content, they are not absolutely identical. 52"96"1'2Thus, prolonged incubation of the high molecular weight form of the purified enzyme with substrates leads to the formation of not one but several low molecular weight forms. 116 A variant of this hypothesis is that NADH dehydrogenase in the membrane contains two "proteins", a flavoprotein (the low molecular weight form of NADH dehydrogenase) and an iron protein, which reoxidizes the former during NADH oxidation in mitochondria."7 It may be noted that the flavoprotein and the iron protein isolated by Hatefi and Stempe1117 do not account for all the polypeptides present in Complex I; 'lz hence, the hypothesis that the Complex I region of the respiratory chain consists of these two "proteins" is contrary to existing data. To quote a recent review,'12 "although catalytically active fragments can be isolated after disruption of the enzyme, their properties are grossly or irreversibly altered. At the present state of knowledge, it is premature or even incorrect to refer to Complex I as a multienzyme complex. It should be more accurately referred to as a multisubunit enzyme." This is, substantially, also the view of the present authors, who have regarded the high molecular weight form of NADH dehydrogenase as resembling closely the particulate Complex I, with a metastable structure in the soluble, purified state, one which may be fragmented in various ways to yield other metastable forms, which have been collectively referred to as "low molecular weight" forms. 52,1,8 One may well wonder why, with the advent of reliable techniques for subunit analysis the relationship of the high molecular weight form to the low molecular weight one and to Complex I has not been settled. Suffice it to say that the complexity of this enzyme seems to have defied even the resolving power of sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis. Thus not only has the polypeptide composition of Complex I given surprisingly different answers in four different laboratories, '12'1'9-'2' although the preparations have been regarded as being '~pure," ,,2 but even the -78,000 molecular weight fragment, relatively easy to obtain in pure form, has been reported to have different subunit composition in different laboratories."2, 1Zl ,is C. Rossi. T. C r e m o n a , J. M. Machinist, and T. P. Singer, J. Biol. Chem. 240, 2364 (1965). 117 y . Hatefi and K. E. Stempel, J. Biol. Chem. 244, 2350 (1969). "~ T. P. Singer, in " C o m p r e h e n s i v e B i o c h e m i s t r y " (M. Florkin and E. H. Stotz, eds.), Vol. 14, p. 127. Elsevier, A m s t e r d a m , 1966. 1,9 R. A. Capaldi, Arch. Biochem. Biophys. 163, 99 (1974). 120 j. F. Hare and F. L. Crane, J. Subcell. Biochem. 3, 1 (1974). 121 E. C. Slater, in " E l e c t r o n Transfer Chains and Oxidative Phosphorylation'" (E. Quagliariello, S. Papa, F. Palmieri, E. C. Slater, and N. Siliprandi, eds.), p. 3. North-Holland Publ., A m s t e r d a m , 1975.

418

FLAVOPROTEINS

[42]

Before closing this overview, it may be appropriate to offer some guidance to those not working in the field in regard to the selection of experimental material for studies on NADH dehydrogenase, perhaps the most complex of all known flavoproteins. In many investigations NADH oxidation may be conveniently studied in ETP, using either ferricyanide or 02 as the terminal acceptor. A useful modification of the original ETP preparation of Crane e t a l . 12z has been published. 98 If a more purified but still particulate enzyme is required, one that retains rotenone-sensitive coenzyme Q reductase activity, Complex I is recommended.'°6 The disadvantages of this preparation are that the preparation is laborious and the yield is low ''2 and that, even in the hands of the original authors, '23 the procedure may yield preparations contaminated with cytochromes.'24 For most studies involving spectrophotometric methods, the high molecular weight form of the soluble enzyme is the preparation of choice. Of the several preparations available the phospholipase extracted enzyme, as described by Ringler e t a l . a8 and modified by Cremona and Kearney 125 is recommended, although the terminal steps in the isolation are not simple. Alternative procedures for isolating the high molecular weight form involve extraction with Triton X-100.126,127 The disadvantage of these procedures is that this detergent modifies the catalytic properties of the enzyme. '28 Another laboratory 1'2 was unable to substantiate the claim '27 that Triton extraction yields rotenone-sensitive coenzyme Q reductase activity. The high molecular weight form has also been extracted with the detergent Lubrol WX,'29 but it is not known whether this procedure modifies the kinetic properties of the enzyme. Although the usefulness of the low molecular weight form of the enzyme is quite limited, since the properties are extensively modified in all preparations, there are numerous acceptable procedures for its isolation. The choice of preparation seems to be arbitrary, depending on the experiences of the particular laboratory. One author 1'2 recommends the resolution of Complex I by chaotropic agents.'3° In the present authors" experience, the application of the heat-acid-ethanol method of Mahler e t a l . ~' to ETP, a fairly readily available source material, also yields a homogeneous preparation in a reproducible manner."5 ,22 F. L. Crane, J. L. Glenn, and D. E. Green, Biochim. Biophys. Acta 22, 475 (1956). ,~3 y. Hatefi, Proc. Natl, Acad. Sci. U.S.A. 60, 733 (1968). 124 M. Gutman, T. P. Singer, H. Beinert, and J. E. Casida, Proc. Natl. Acad. Sci. U.S.A. 65, 763 (1970). ,25 T. Cremona and E. B. Kearney, J. Biol. Chem. 239, 2328 (1964). 126 Z. Kaniuga, in "Flavins and Flavoproteins" (H. Kamin, ed,), p. 649. Univ. Park Press, Baltimore, Maryland, 1971. ,2~ R. F. Baugh and T. E. King, Biochem. Biophys. Res. Commun. 49, 1165 (1972). ,28 M. Gutman, Physiol. Chem. Phys. 2, 9 (1970). ,29 p. K. Huang and R. L. Pharo, Biochim. Biophys. Acta 245, 240 (1971). la0 K. A. Davis and Y. Hatefi, Biochemistr), 18, 3355 (1969).