- Email: [email protected]
[13] Flavodoxins
188
[13]
DISSIMILATORY SULFATE REDUCTION
for recognition. As a final remark, it is remarkable to notice that the interconversion process is a sequence of redox and coordination events: [3Fe-4S] 1+
e
~ [3Fe-4S] ° E0 (3Fe)
Fe(2 + )
~ [4Fe-4S] 2+
e
~ [4Fe-4S] 1+ E0 (4Fe)
The fact that an increase in affinity for iron is detected when [3Fe-4S] is reduced, 69,79and the order ofredox potential observed [E0(3Fe) > E0(4Fe)], led to the conclusion that uptake or loss of an iron site from the cluster is related to iron concentration and redox potential. Also, the observation that an additional ligand is required (in general an extra cysteine) when the fourth site of the cube is filled makes the iron atom trigger a redoxlinked conformational change. The inorganic element, iron, is a sensor, linked to the redox state of the cell. 79'82 These considerations motivate the search for the physiological role of the [3Fe-4S] centers and further studies on the significance of the cluster interconversions observed. The SRB ferredoxins and the metabolic pathways involved seem to be an adequate system for gathering answers to these questions.
[13] F l a v o d o x i n s By J A C Q U E S
VERVOORT, DIRK HEERING,
and
WILLEM
SJAAK PEELEN,
VAN BERKEL
Introduction Flavodoxins are a group of relatively small monomeric flavoproteins ( M r 15,000-22,000) containing a single molecule of noncovalently bound
ribofavin 5'-phosphate (FMN) (Fig. 1). This cofactor functions as a redox center and is involved in electron transfer, which is the main biological function of flavodoxins. Flavodoxins can be found in many prokaryotes and also in some eukaryotic algae. In Desuifovibrio spp. the flavodoxins have been suggested to be involved in (1) the electron transport from hydrogenase to the sulfite-reducing system, using molecular hydrogen as the electron donor, or (2) as electron carrier from the pyruvate phosphoroclastic system toward the sulfite-reducing system, using pyruvate as electron donor.l I J.-H. K i m a n d J. M. Akagi, J. Bacteriol. 163, 472 (1985).
METHODS IN ENZYMOLOGY,VOL. 243
Copyright © 1994by AcademicPress, Inc. All rights of reproduction in any form reserved.
[ 13]
FLAVODOXINS
189
5' C,H,OPO,H, I 4' CHOH I 3' CHOH
I
2' CHOH
I
1' OH, I -N
H,C
,,.,,"~6
H.C-
~
5~
0~
N
O
4 , ~ HN
"N"
0 FIO. 1. Structure of riboflavin Y-phosphate (FMN).
In the p r e s e n c e of iron in the culture medium of sulfate-reducing bacteria, h o w e v e r , the biosynthesis of flavodoxins is suppressed at the expense of ferredoxin. 2'3 F l a v o d o x i n s s e e m to be able to take o v e r the role of ferredoxins as electron d o n o r in the sulfite-reducing system. Purification T h e purification of D e s u l f o o i b r i o flavodoxins can be done according to the p r o c e d u r e s of L e Gall and Hatchikian, 4 M o u r a et al.,5 and Ifie et al. 6 Reagents and Materials
D E A E - 5 2 cellulose S e p h a d e x G-50 Tris-HC1 buffer (50 raM), p H 7.6 All steps are carried out at 4 ° unless specified otherwise. The D e s u l f o o i b r i o cells are suspended in 50 m M Tfis-HCl buffer (about 1 g of cells/ml buffer). The cells are subsequently disrupted by a French press and centrifuged at 30,000 g for 30 min. T h e supernatant is passed o v e r a DEAE-52 cellulose 2 H. D. Peck, Jr., and J. LeGall, Philos. Trans. R. Soc. London B. 298, 443 (1982). 3 S. G. Mayhew and M. L. Ludwig, Enzyme 12, 57 (1975). 4 j. LeGall and E. C. Hatchikian, C.R. Acad. Sci. Paris 264, 2580 0967). 5 I. Moura, J. J. G. Moura, M. Brusch, and J. LeGall, Biochim. Biophys. Acta 591, 1 0980). 6 K. Irie, K. Kobayashi, M. Kobayashi, and M. Ishimoto, J. Biochem. (Tokyo) 73, 353 (1973).
190
DISSIMILATORY SULFATE REDUCTION
[13]
column, equilibrated with 50 mM Tris-HCl buffer. The bound flavodoxin is eluted from the column with 0.15 M Tris-HC1 buffer, 0.4 M KC1, pH 7.6. This fraction is desalted over a Sephadex G-50 column (in 50 mM Tris-HCl, pH 7.6) or alternatively dialyzed against 50 mM Tris-HCl buffer, pH 7.6. The protein is then brought onto a DEAE-52 cellulose column (volume > 150 ml) equilibrated with Tris-HC1 buffer (50 mM, pH 7.6) and the flavodoxin is eluted from the column by a linear gradient of Tris-HCl (50 mM to 0.5 M). The flavodoxin fraction elutes at 0.30-0.35 M TrisHCI. As the flavodoxin fraction can still contain other protein components (mainly rubredoxin) this fraction is dialyzed against 50 mM Tris-HCl buffer (pH 7.6) and brought onto a second DEAE-52 cellulose column, which is then eluted with a linear gradient of Tris-HCl (0.25 to 0.40 M, pH 7.6). Redox Potentials Flavodoxins can occur in three redox states (Fig. 2). Of particular physiological importance3 is the transition between the one electron-reR
I
H3cWN'Ny°
OXIDIZED
N"
H+,e.llO2 0 RI FI3C~N~N~O SEMIQUINONE
eltO'
o
R I
HaC~NvN.,,~O HYDROQUINONE
0 FIG. 2. The structures of oxidized (quinone), one electron-reduced (semiquinone), and two electron-reduced (hydroquinone) flavin.
[13]
FLAVODOXINS
191
TABLE I REDOX POTENTIALSOF FLAVODOXINS(IN MILLIVOLTS)a
Desulfovibrio vulgaris CIostridium beijerinckii MP Megasphaera elsdenii Anabaena variabilis Anacystis nidulans Azotobacter vinelandii Azotobacter chroococcum Escherichia coli Klebsiella pneumoniae Riboflavin 5'-phosphate (FMN)
Ez
El
Ref.
- 143 - 92 - 115 - 195 - 221 - 165 - 115 -240 - 158 -314
- 435 - 399 - 372 - 390 - 447 -458 - 520 -410 -412 - 124
7 8 9 10 11 12 13 14 13 15
a E1 , the transition from semiquinone to the hydroquinone form; E 2, the transition from the oxidized to the semiquinone form. All values indicated are at pH 7.
duced state and the two electron-reduced state. The redox potentials of this t r a n s i t i o n a r e a m o n g t h e l o w e s t o b s e r v e d in n a t u r e , r a n g i n g f r o m - 3 0 5 to - 5 2 0 m V ( T a b l e I). 7-15 R e d u c t i o n o f o x i d i z e d f l a v o d o x i n to t h e s e m i q u i n o n e f o r m c a n e a s i l y be achieved by the addition of (chemical) reducing agents. However, o w i n g to t h e l o w r e d o x p o t e n t i a l o f t h e s e m i q u i n o n e - h y d r o q u i n o n e t r a n s i t i o n ( h i s t o r i c a l l y c a l l e d E 0 c o m p l e t e r e d u c t i o n o f D e s u l f o v i b r i o vulgaris f l a v o d o x i n is difficult to a c h i e v e a n d d e p e n d s o n t h e r e l a t i v e r e d o x p o t e n tials o f t h e e l e c t r o n a c c e p t o r ( f l a v o d o x i n ) a n d e l e c t r o n d o n o r . T h e m o s t w i d e l y u s e d e l e c t r o n d o n o r is d i t h i o n i t e . A t l o w c o n c e n t r a tions of flavodoxin (<0.5 mM) reduction can be accomplished rather easily by the near stoichiometric addition of a freshly prepared dithionite solution in p h o s p h a t e b u f f e r , p H 7 ( 1 0 0 - 2 0 0 m g / m l ) . I t is a d v i s a b l e to flush t h e buffer with (oxygen-free) argon before addition of dithionite. However, 7 G. P. Curley, M. C. Carr, P. A. O'Farell, S. G. Mayhew, and G. Voordouw, in "Flavins and Flavoproteins 1990" (B. Curti, S. Ronchi, and G. Zanetti, eds.), p. 429. de Gruyter, Berlin, 1991. 8 S. G. Mayhew, Biochim. Biophys. Acta 235, 276 (1971). 9 S. G. Mayhew, G. P. Foust, and V. Massey, J. Biol. Chem. 244, 803 (1969). l0 M. F. Fillat, G. Sandmann, and C. Gomez-Moreno, Bioehem. Biophys. Res. Commun. 1040, 301 (1990). 11 B. Entsch and R. M. Smillie, Arch. Biochem. Biophys. 151, 378 (1972). 12M. F. Taylor, W. H. Boylan, and D. E. Edmondson, Biochemistry 29, 6911 (1990). 13j. Deistung and R. N. F. Thorneley, Biochem. J. 239, 69 (1986). 14 H. Vetter, Jr., and J. Knappe, Hoppe-Seyer's Z. Physiol. Chem. 352, 433 (1971). 15R. F. Anderson, Biochim. Biophys. Acta 772, 158 (1983).
192
DISSIMILATORY SULFATE REDUCTION
[13]
at high flavodoxin concentrations (>0,5 mM) reduction to a 100% hydroquinone form is virtually impossible to achieve at pH values below 7,5 and at ionic strengths below 100 mM. J6At high concentrations of flavodoxins (> 1 mM) complete reduction to the hydroquinone form by dithionite (at 200-mg/ml stock solution) can be achieved when keeping the pH of the sample above pH 8. (The flavodoxin sample should be oxygen free, as reduction of the residual oxygen by dithionite gives rise to pH changes that make reduction more difficult.) The contamination of sodium dithionite with small amounts of (bi)sulfite may cause oxidation of flavodoxin. 16 Therefore it is essential to use the highest purity of dithionite available. Reduction of flavodoxins can also be accomplished by illumination (50- to 100-W tungsten lamp) of an anaerobic solution of flavodoxin in the presence of ethylenediaminetetraacetic acid (EDTA) (50 mM) and of a catalytic amount of 5-deazariboflavin. The reduction to the semiquinone form is completed typically within I hr. Continued illumination (several hours) yields the hydroquinone. A major advantage over reduction by dithionite is that by using EDTA and light no spectral disturbance occurs in the region of 280-380 nm.17 The flavodoxin redox potentials can be determined by controlled titration with dithionite and subsequent measurement of the concentration of the semiquinone radical by electron paramagnetic resonance (EPR). 1a,19 Typically a deaerated and argon-flushed buffered solution of 20 to 50/xM flavodoxin and a l0/xM series of mediator dyes is titrated with dithionite in buffer under a constant flow of argon. The potential of the solution is measured between a platinum electrode and a calomel or Ag/AgC1 reference electrode. After each addition of dithionite, and after allowing the solution to equilibrate, a sample is anaerobically injected into an EPR tube and frozen in liquid nitrogen. The potential range of the mediators must be sufficient to buffer the potential at any point of the titration. The use of mediators accepting two electrons is recommended to avoid interference of mediator radical signals with the protein semiquinone signal. However, for the titration of E~, no two-electron mediators are available. Mediators accepting one electron can be used if the concentrations of these mediators are low with respect to the protein and the sharp mediator radical signal can be subtracted from the broader protein signal. The relative concentration of semiquinone as determined from the EPR spectra (corrected for dilution) is plotted versus the potential and fitted to the Nernst equation to determine the redox potentials. 16 S. G. Mayhew, Eur. J. Biochem. 85, 535 (1978). 17 V. Massey and P. Hemmerich, Biochemistry 17, 9 (1978). 18 G. S. Wilson, this series, Vol. 54, p. 396. 19 p. L. Dutton, this series, Vol. 54, p. 411.
[13]
FLAVODOXINS
193
Alternatively, a potentiometric titration can be performed by measuring the concentrations of quinone, semiquinone, and hydroquinone spectrophotometricaUy. The reduction is performed by adding dithionite (E2), by hydrogen in the presence of hydrogenase (E0, or by photoreduction (E2 and E0. The potential of the solution can be calculated from the amount of reductant added (or partial hydrogen pressure) or from the redox state of a reference mediator dye, or may be directly measured as described. 17-zl The quinone-semiquinone potential can also be determined by the spectrophotometric xanthine/xanthine oxidase titration described by Mass e y . 22 The method uses the low potential of the xanthine/ureate couple (-350 mV at pH 7) together with the ability of xanthine oxidase and flavoproteins to exchange electrons with redox dyes. Neither xanthine nor ureate interfere in the near ultraviolet (UV)-visible range. Typically, a buffered solution of 10 to 20 ~M flavodoxin, 200-300/xM xanthine, a redox dye with a potential within 30 mV from the unknown, and 2/zM methyl or benzyl viologen is placed in an anaerobic cuvette, deaerated, and flushed with argon. It is preferable to use a redox dye with a measurable difference in absorption between the oxidized and reduced form at an isosbestic point of the flavodoxin. At t = 0 an anaerobic solution of xanthine oxidase is added and spectra are recorded. The amount of xanthine oxidase must be low (10-50 nM) to ensure equilibrium conditions. The complete reduction is typically completed within 1 to 2 hr and about 50-100 spectra are recorded. The potential of the flavodoxin can be determined by plotting its log(ox/red) value versus the log(ox/red) value of the redox dye. The slope of the plot is unity when the two couples involve the same number of electrons. The difference between the potentials can be calculated (using the Nernst equation) from the log(ox/red) value of the flavodoxin at the point where the log(ox/red) value of the dye is zero. As an alternative for the titrative determination of redox potentials, direct electrochemistry of redox proteins has become feasible over the last 10 years.Z3-31Direct electrochemistry of flavodoxins has been reported 20 M. Dubourdieu, J. LeGall, and V. Favaudon, Biochim. Biophys. Acta 376, 519 (1975). 21 S. G. Mayhew and V. Massey, Biochim. Biophys. Acta 315, 181 (1973). 22 V. Massey, "Proceedings of the Tenth International Symposium on Flavins and Flavoproteins," p. 59. de Gruyter, Berlin, 1991. 23 C. Van Dijk, J. W. Van Leeuwen, and C. Veeger, Bioelectrochem. Bioenerg. 9, 743 (1982). 24 W. R. Hagen, Eur. J. Biochem. 182, 523 (1989). 25 L. H. Guo, H. A. O. Hill, G. A. Lawrence, G. S. Sanghera, and D. J. Hopper, J. Electroanal. Chem. 266, 379 (1989). 26 p. Bianco, J. Haladjian, A. Manjaoui, and M. Bruschi, Electrochim. Acta 33, 745 (1988). 27 F. A. Armstrong, J. N. Butt, and A. Sucheta, this series, Vol. 227, p. 479. 28 F. A. Armstrong, H. A. O. Hill, B. N. Oliver, and N. J. Walton, J. Am. Chem. Soc. 106, 921 (1984).
194
DISSIMILATORY SULFATE REDUCTION
[13]
by Van Dijk, 23 Armstrong, Hill, and co-workers. 27,28 Van Dijk, 23 using differential and normal pulse polarography, determined the quinone/semiquinone ( - 114 mV, pH 7.4) and semiquinone/hydroquinone ( - 392 mV, pH 7.4) couples of M. elsdenii flavodoxin adsorped at the mercury electrode. At the pyrolytic graphite electrode, Armstrong et al. 2s only found the semiquinone/hydroquinone couple of M. elsdenii flavodoxin (-318 mV, pH 5.0) using square wave voltammetry and in the presence of MgCI/ and Cr(NH3)63+. Bianco et al. 26 determined the semiquinone/hydroquinone couple of D. vulgaris (Hildenborough) flavodoxin (-430 mV, pH 7.6) at the pyrolytic graphite electrode using differential pulse voltammetry and cyclic voltammetry. Cyclic voltammetry of the flavodoxins isolated from Azotobacter choococcum at a polished edge-plane graphite electrode has been performed29 in the presence of the cationic aminoglycoside neomycin. The semiquinone-hydroquinone potentials of two distinct flavodoxins were found (-305 and -520 mV at pH 7.4). Our group has investigated the electrochemistry of D. vulgaris Hildenborough. 3°,3~ In the classic three-electrode setup and semiinfinite diffusion, a bulk solution of about 1 mEand a protein concentration of 100/zM is required. In the setup developed in our laboratory the volume of the bulk is reduced to less than 20/xl with full retention of semiinfinite diffusion down to a scan rate of 1 mV/sec. ~4 The working electrode is a dismountable inverted disk of glassy carbon (15-ram diameter and 2-mm height, type V25; obtained from Le Carbon Loraine, Paris). Prior to each electrochemical measurement the disk is polished firmly with polishing cloth with 6-/~m diamond lapping compound (Engis, Ltd., Kent, England), rinsed thoroughly with water, and dried. To activate the electrode it is exposed for 30 sec to a methane flame from a Bunsen burner. The polished working surface is never in direct contact with the flame, and if any inhomogeneity is detected on the surface the polishing and glowing are repeated. The counterelectrode is a microplatinum electrode (P-1312; Radiometer) and the reference electrode is a saturated calomel electrode (K-401; Radiometer). After mounting the electrodes the cell is flushed with wet argon. A deaerated, argon-flushed sample is then transferred with a gas-tight Hamilton syringe to the tip of the reference electrode and the working electrode holder is pushed up until the shape of the droplet is approximately cylindrical. The electrodes are connected to an Autolab 10 potentiostat (Eco Chemie) controlled by GPES software (version 2.0) (Eco Chemie) on a PC. 29 S. Bagby, P. D. Barker, H. A. O. Hill, G. S. Sanghera, B. Dunbar, G. A. Ashby, R. R. Eady, and R. N. F. Thorneley, Biochem. J. 277, 313 (1991). 30 H. A. Heering and W. R. Hagen, J. Inorg. Biochem. 51, 25 (1993). 31 H. A. Heering and W. R. Hagen, unpublished results (1994).
[ 13]
FLAVODOXINS
-66o
'
-4'o0
-2bo
195
b
'
260
Em (mY)
FIG. 3. Staircase cyclic voltammogram (20 mV/sec) of 6 /zl of 0.14 mM D. oulgaris flavodoxin in 20 mM potassium phosphate, pH 7.0: before and after addition of 0.5 pJ of 50 mM neomycin.
Staircase cyclic voltammetry (SCV) was done with steps of 2.44 inV. Differential pulse polarography (DPP) was done in both directions (after a 60-sec equilibration at the starting potential) with steps of 2.44 mV and 250 msec, modulated by pulses of 20 mV and 100 msec (Fig. 3). The experiments were performed at a temperature of 22 - 1° and the potentials have been recalculated with respect to the normal hydrogen electrode (NHE), using the potential of - 2 4 6 mV (NHE) of the saturated calomel electrode (SCE). The promotor neomycin B (Sigma, St. Louis, MO) is added from a 50 mM solution, titrated to pH 7.0 with NaOH. The pH dependence is measured with SCV and DPP. The SCV measurements are performed on droplets of 5 ~1 of 0.11 mM flavodoxin and 3.3 mM neomycin in 20 mM citrate, 20 mM bistrispropane, and 20 mM CAPS, titrated with HCI or NaOH and adjusted to ionic strength/z = 0.2 with NaC1. The DPP measurements were performed on droplets of 5 /xl 0.13 mM flavodoxin and 3 mM neomycin in the same buffer. In all experiments the fully oxidized flavodoxin (as determined spectroscopically) was used. As in the experiments of Armstrong e t a / . 27'28 and of Bagby et a1.,29 the first reduction (quinone to semiquinone) is not detectable at the electrode and the second reduction step is detected only after addition of a cationic promotor such as neomycin. The cations are believed to form a
196
DISSIMILATORY SULFATE REDUCTION
[13]
O. Xin: p K a = 4 . 8
DPP
-100
V
'-E -iZ r/)
-200
>
E E -300
=-'~--m- m,,~.~ m= .,.. 0H
m
0
1o
Fxo. 4. pH dependence of the midpoint potentials of FMN (adsorbed at the electrode) and of D. vulgaris flavodoxin El in the presence of neomycin and at constant ionic strength (/~ = 0.2). (rq) DPP; (1) CV.
bridge between the negative charges of both the electrode and flavodoxin (pl = 3.6). Without neomycin (at pH 7.0) only one redox couple is found at a potential of -217 mV (NHE) with a peak current proportional to the scan rate. This response is caused by FMN, dissociated from the protein and adsorbed onto the electrode. The potential and the PKa of 6.6 are equal to those measured for free FMN in solution. After addition of neomycin one additional response is observed with a peak current proportional to the square root of the scan rate and the characteristics of a oneelectron transition. The potential of -409 mV (NHE) is near the chemically determined potential of the semiquinone-to-hydroquinone reduction. 2°'32 The observed PKa of 4.8 is, however, much lower than the reported value of 6.620 to 6.8. 32 This difference between the pK a at the electrode and the one reported for the chemically reduced flavodoxin might be caused by a conformational change near the electrode. The measured pKa of 4.8 can be due to the altered environment of the amino acid residue responsible for the redox-linked PKa. Alternatively, the residue might be blocked or no longer in the vicinity of the isoalloxazine. The measured pK a of 4.8 can be due to protonation of the isoalloxazine at N-1. See Fig. 4. The midpoint potential of the quinone to semiquinone reduction (as determined by EPR titration with dithionite) is -113 mV (NHE) at pH 7.0. This value is in agreement with reported potentials. 2°'32 The absence of the quinone to semiquinone couple in the voltammograms can be ex32 G. P. Curley, M. C. Carr, S. G. Mayhew, and G. Voordouw, Fur. J. Biochem. 202, 1091 (1991).
[ 13]
FLAVODOXINS
197
plained 3°'31 by the known fast comproportionation mechanism of one fully reduced and one fully oxidized flavodoxin to two semiquinone flavodoxin molecules 2° in combination with a very slow (virtually not occurring) reduction of the oxidized to the semiquinone form. 21 By consequence, only a few molecules (catalytic amount) of semiquinone are sufficient to generate equilibrium concentrations of semiquinone and hydroquinone near the electrode surface and the development of the voltammogram without a visible first redox couple. A catalytic amount of free FMN may also act as a mediator to generate the first molecules of semiquinone, Amino Acid Sequences and Structure The amino acid sequences of several flavodoxins are known (Table II). The homology between these sequences ranges from 20 to 40%. As alignment solely on the basis of sequences is known to be difficult, structural alignment was investigated using the structures of the flavodoxins from Chondrus crispus, D. vulgaris, and Clostridium beijerinckii. The structurally conserved regions were then determined. On the basis of these conserved regions, the alignment of the other flavodoxin sequences was performed using the Clustal V alignment program. 33 Conserved residues are marked in bold in Table I I . 34-46 As is evident from this alignment only five residues are totally conserved. The first four of these residues are embedded close to each other in a hydrophobic pocket in the protein. These four residues may either be of importance for electron transfer processes or may be important in the folding process of flavodoxins. As can be inferred from the amino acid sequences, flavodoxins are acidic proteins. Their net negative charges ranges from - 1 0 to -20, and their isoelectric points are situated around pH 4. The low isoelectric points, high 33 D. G. Higgins and P. M. Sharp, Gene 73, 237 (1988). 34 M. Dubourdieu and J. L. Fox, J. Biol. Chem. 252, 1453 (1977). 35 L. R. Helms and R. P. Swenson, Biochim. Biophys. Acta 1089, 417 (1991). 36 L. R. Helms, G. D. Krey, and R. P. Swenson, Biochem. Biophys. Res. Commun. 168, 809 (1990). 37 M. Tanaka, M. Haniu, K. T. Yasunobu, and S. G. Mayhew, J. Biol. Chem. 249, 4393 (1974). 38 D. Santangelo, D. T. Jones, and D. R. Woods, J. Bacteriol. 173, 1088 (1991). 39 M. Tanaka, M. Haniu, K. T. Yasunobu, S. G. Mayhew, and V. Massey, J. Biol. Chem. 249, 4397 (1974). 4o S. Wakabayashi, T. Kimura, K. Fukuyama, H. Matsubara, and L. J. Rogers, Biochem. J. 263, 981 (1989). 41 K. G. Leonhardt and N. A. Straus, Nucleic Acids Res. 17, 4384 (1989). 42 D. E. Laudebach, M. E. Reith, and N. A. Straus, J. Bacteriol. 170, 258 (1988). 43 L. T. Bennett, M. R. Jacobson, and D. R. Dean, J. Biol. Chem. 263, 1364 (1988). 44 C. Osborne, L. M. Chen, and R. G. Matthews, J. Bacteriol. 173, 1729 (1991). 45 W. Arnold, A. Rump, W. Klipp, U. B. Priefer, and A. J. Puehler, J. Mol. Biol. 203, 715 (1988). 46 y . Jouanneau, P. Richard, and C. Grabau, Nucleic Acids Res. 18, 5284 (1990).
198
DISSIMILATORY
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. .
,
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.
[13]
~
m
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o o o Z ~ u , ,..l [-.-,
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[ 13]
FLAVODOXINS
199
al<
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~ < ~ m ~ m Z Z Z Z m
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,
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<
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u.lm~ u a ~ ,.2
~'~
~ ~
~
2 <
°~
200
DISSIMILATORY SULFATE REDUCTION
[13]
stability, and high charge density make it possible to prepare flavodoxin solutions of up to 400 mg/ml. At these high concentrations no aggregation phenomena can be observed as is evident from ~H nuclear magnetic resonance (NMR) studies at these concentrations. 47'48 This property, in combination with their low molecular weight, makes flavodoxins good candidates for structure determination using multidimensional NMR techniques. From such multidimensional NMR studies it became clear that the solution and crystal structures of D. vulgaris flavodoxin are virtually identical. 48 The tertiary structures of all flavodoxins share a common polypeptide fold consisting of a central parallel fl sheet with five strands surrounded on both sites by helices. The FMN is strongly bound via H bonds on one side of the protein with the isoalloxazine moiety exposed to the solvent. The loop regions that surround the isoalloxazine ring (residues 59-64 and 94-104 in D. vulgaris flavodoxin) have hardly any conserved sequences. Nevertheless, in general the isoalloxazine ring seems to be sandwiched between two aromatic residues (W60 and Y98 in D. vulgaris flavodoxin). In some flavodoxins one of the two aromatic residues is replaced by a hydrophobic residue (M, L, or I). It appears, on comparison of the known structures and amino acid sequences (Table II), that the isoalloxazine ring needs to be shielded from the solvent by these residues for the establishment of the low redox potentials. 49 The reader is referred to detailed reviews on the three-dimensional structure characteristics of flavodoxins.50
Apoflavodoxin Preparation Apoflavodoxin can be prepared according to the following procedure (this procedure contains slight modifications of the original procedure of Wassink and Mayhew 51).
Procedure 1. Cold trichloroacetic acid (TCA)(T = 4°) at a concentration of 40% (w/v) is slowly added to a cold flavodoxin solution (concentration 0.2-0.4 mM, 4°) in 0.1 M phosphate buffer (pH 7-8)-0.3 mM EDTA, to give a final concentration of 5% TCA. The flavodoxin solution should preferably be shielded from light. After the addition of TCA the solution 47 S. S. Wijmenga and C. P. M. van Mierlo, Eur. J. Biochem. 195, 807 (1991). 48 S. Peelen, J. Vervoort, and J. LeGall, unpublished results (1994). 49 j. Vervoort, Curt. Opin. Struct. Biol. 1, 889 (1991). 5o M. L. Ludwig and C. L. Luschinsky, in "Chemistry and Biochemistry ofFlavoenzymes" (F. MOiler, ed.), Vol. 3, p. 427. CRC Press, Boca Raton, Florida, 1991. 5z j. H. Wassink and S. G. Mayhew, Anal. Biochem. 68, 609 (1975).
[13]
FLAVODOX|NS
201
is centrifuged at 10,000 g for 10 min. The white precipitate can be dissolved in 0.1 M potassium phosphate-0.3 mM EDTA (pH 7-8). As the procedure does not give 100% apoprotein in one run, it is advisable, when necessary, to repeat the procedure. The apoprotein prepared in this way is stable for a long period: more than 1 month at 4° up to 1 week at room temperature. Procedure 2. A milder procedure for apoflavodoxin preparation is as follows: Dialyze 0.2-0.4 mM flavodoxin in 0.1 M potas sium phosphate-0.3 mM EDTA (pH 7-8) four times against 250 ml of 2 M KBr in 0.1 M sodium acetate buffer, pH 3.9, with 0.3 mM EDTA (total time, 48 hr). The precipitate can be redissolved in 0.1 M potassium phosphate (pH 7.0)-0.3 mM EDTA. The disadvantage of this procedure is the long time span needed to prepare the apoprotein. The apopreparation procedure does not lead to irreversible modifications of the apoprotein. 31p and 1H NMR studies of flavodoxins from D. vulgaris and also from M. elsdenii, before (native) and after apopreparation followed by reconstitution with FMN, give identical spectra. From this the firm conclusion can be drawn that the recombined protein (apoprotein recombined with FMN) and the native protein have the same conformation. Reconstitution of Holoflavodoxin from Apoprotein and Ribofavin 5'-Phosphate A wealth of information is available about the reconstitution of holoflavodoxin from its constituents, the apoprotein and prosthetic group. For details about the thermodynamics and kinetics of flavin binding the reader is referred to the review of Mayhew and Ludwig) Apoflavodoxins from Clostridium species and M. elsdenii are specific for binding of flavins at the FMN level) 2,53 The apoflavodoxins from Azotobacter vinelandii and D. vulgaris form tight complexes not only with FMN but also with riboflavin and lumiflavin analogs. 54,55 Apoflavodoxins tightly bind FMN at neutral pH. For both M. elsdenii and D. vulgaris flavodoxin, the dissociation constant (Kd) of the complex is in the range of 10-10 M.52,56 Stabilization of the interaction between apoprotein and prosthetic group is de52 S. G. Mayhew, Biochim. Biophys. Acta 235, 289 (1971). 53 j. A. D'Anna and G. Tollin, Biochemistry 11, 1073 (1972). 54 D. E. Edmondson and G. Tollin, Biochemistry 10, 113 (1971). 55 D. E. Edmondson and G. Tollin, Biochemistry 10, 133 (1971). 56 j. Vervoort, W. J. H. van Berkel, S. G. Mayhew, F. Mt~ller, A. Bacher, P. Nielsen, and J. Legall, Eur. J. Biochem. 161, 749 (1986).
202
DISSIMILATORY SULFATE REDUCTION
[13]
creased at pH values below 5 and is also dependent on the ionic strength and type of anions present in solution. 57 Binding of FMN at neutral pH is rapid and results in almost complete quenching of flavin fluorescence?2 This property has been used with the apoflavodoxin from M. elsdenii to assay for FMN and FAD in mixtures and to analyze commercial FMN preparations for their content. 51 Commercial FMN preparations contain 25-30% of fluorescent impurities. 51,58 Synthesis of FMN by chemical phosphorylation of riboflavin yields 4'-FMN as a main contaminant. 59 By 31p NMR it was shown that commercial FMN also contains considerable amounts of the 3' and 2' isomers and other phosphorus-containing compounds. 6° The impurities (including riboflavin) and 5'-FMN are conveniently separated by reversedphase high-performance liquid chromatography (HPLC) 61and preparative amounts of each compound can be obtained in pure form. 62'63On the basis of various chemical properties and enzymatic hydrolysis, the structures of the phosphorylated impurities have been assigned t o 61'62 riboflavin 4'-phosphate (4'-FMN), riboflavin 3'-phosphate (Y-FMN), riboflavin 2'-phosphate (2'-FMN), riboflavin 4',5'-bisphosphate (4',5'-FBP), riboflavin 3',5'-bisphosphate (3 ',5'-FBP), riboflavin Y,4'-bisphosphate (3',4'FBP), riboflavin 2',5'-bisphosphate (2',5'-FBP), riboflavin 2',4'-bisphosphate (2' ,4'-FBP) and riboflavin 4',5'-cyclophosphate (4',5'-FCP), respectively. The occurrence of these flavin isomers is explained by the acidcatalyzed migration of the phosphoric acid groups in both the riboflavin mono- and bisphosphates. 59'62 At neutral pH, the isomerization reactions are slow as compared to hydrolysis of the phosphoester bond. 6z For a long time it was thought that apoflavodoxin is highly specific for 5'-FMN. 5~Separation of the isomeric riboflavin mono- and bisphosphates, however, revealed that apoflavodoxin from M. elsdenii also binds 3',5'FBP tightly. 6~This compound makes up 2% of the total amount of commercial FMN. 62Megasphaera elsdenii apoflavodoxin reconstituted with Y,5'FBP is fully active as an electron carrier by transferring reduction equivalents from H2 via hydrogenase to metronidazole.6~ This suggested that the additional phosphate group does not influence the properties of the complex. A more thorough kinetic, thermodynamic, and spectral analysis of the complexes of 3',5'-FBP and the apoflavodoxins from M. elsdenii 57 R. Gast, B. E. Valk, F. Mfiller, S. G. Mayhew, and C. Veeger, Biochim. Biophys. Acta 446, 463 (1976). 58 V. Massey and B. E. P. Swoboda, Biochem. Z. 339, 474 (1963). 59 G. Scola-Nagelschneider and P. Hemmerich, Eur. J, Biochem. 66, 567 (1976). 6o C. T. W. Moonen and F. MOller, Biochemistry 21, 408 (1982). 61 p. Nielsen, P. Rauschenbach, and A. Bacher, Anal. Biochem. 130, 359 (1983). 62 p. Nielsen, J. Harksen, and A. Bacher, Eur. J. Biochem. 152, 465 (1985). 63 p. Nielsen, P. Rauschenbacher, and A. Bacher, this series, Vol. 122, p. 209.
[14]
RUBREDOXIN IN CRYSTALLINE STATE
203
and D. vulgaris confirmed this idea. 56Both apoflavodoxins bind 3',5'-FBP somewhat more weakly than 5'-FMN owing to slower association rate constants. The redox potentials of the artificial complex, however, are similar to the values reported for the native protein. 31p NMR experiments revealed that the 3'-phosphate group is accessible for Mn 2÷ and indicates that this group is located close to the protein surface. 56 This is in sharp contrast with the 5'-phosphate group, which is dianionic 6° and buried in both proteins. The 5'-phosphate group therefore determines the specificity of flavin binding, whereas introduction of the 3'-phosphate group only slightly influences the protein conformation. The latter conclusion is in full accordance with results obtained from the crystal structure of D. vulgaris flavodoxin as well as ~H NMR results, which show that the 3'-OH of the ribityl side chain is oriented toward bulk solvent. 48,64 64 W. Watt, A. Tulinsky, R. P. Swenson, and K. D. Wautenpaugh, J. Mol. Biol. 218, 195 (1991).
[14] R u b r e d o x i n in C r y s t a l l i n e S t a t e By
LARRY C . SIEKER, RONALD
E.
STENKAMP,
and
JEAN L E G A L L
General Remarks Rubredoxin (Rd) is one of the simplest of iron proteins and has been found, thus far, only in certain microorganisms. 1 The initial report and characterization of a rubredoxin was done by Lovenberg and Sobel in 1965. 2 Rubredoxins are composed of 45 to 54 amino acid residues with molecular weights ranging from 5000 to 6000 and contain 1 iron atom liganded by 4 cysteine residues. The iron center can be reversibly reduced at a redox potential near 0 inV.1 Although many rubredoxins have been detected and isolated from a variety of bacteria, only 13 of the rubredoxins have had amino acid sequences determined. Figure 1 shows the amino acid sequence alignment of the 13 rubredoxins. Because this chapter is primarily directed to the sulfate-reducing bacteria we have chosen to divide these rubredoxins into three categories. Figure la lists the Rds from the sulfate-reducing Desulfooibrio species, Fig. lb shows the Rds from a mixed assortment of bacteria, and Fig. lc contains the thermophilic Rds. i T. G . Spiro, "Iron-Sulfur Proteins." Wiley (Interscience), New York, 1982. 2 W. Lovenberg and B. E. Sobel, Proc. Natl. Acad. Sci. U.S.A. 54, 193 (1965).
METHODS IN ENZYMOLOGY,VOL. 243
Copyright© 1994by AcademicPress, Inc. All rightsof reproductionin any formreserved.
[13]
DISSIMILATORY SULFATE REDUCTION
for recognition. As a final remark, it is remarkable to notice that the interconversion process is a sequence of redox and coordination events: [3Fe-4S] 1+
e
~ [3Fe-4S] ° E0 (3Fe)
Fe(2 + )
~ [4Fe-4S] 2+
e
~ [4Fe-4S] 1+ E0 (4Fe)
The fact that an increase in affinity for iron is detected when [3Fe-4S] is reduced, 69,79and the order ofredox potential observed [E0(3Fe) > E0(4Fe)], led to the conclusion that uptake or loss of an iron site from the cluster is related to iron concentration and redox potential. Also, the observation that an additional ligand is required (in general an extra cysteine) when the fourth site of the cube is filled makes the iron atom trigger a redoxlinked conformational change. The inorganic element, iron, is a sensor, linked to the redox state of the cell. 79'82 These considerations motivate the search for the physiological role of the [3Fe-4S] centers and further studies on the significance of the cluster interconversions observed. The SRB ferredoxins and the metabolic pathways involved seem to be an adequate system for gathering answers to these questions.
[13] F l a v o d o x i n s By J A C Q U E S
VERVOORT, DIRK HEERING,
and
WILLEM
SJAAK PEELEN,
VAN BERKEL
Introduction Flavodoxins are a group of relatively small monomeric flavoproteins ( M r 15,000-22,000) containing a single molecule of noncovalently bound
ribofavin 5'-phosphate (FMN) (Fig. 1). This cofactor functions as a redox center and is involved in electron transfer, which is the main biological function of flavodoxins. Flavodoxins can be found in many prokaryotes and also in some eukaryotic algae. In Desuifovibrio spp. the flavodoxins have been suggested to be involved in (1) the electron transport from hydrogenase to the sulfite-reducing system, using molecular hydrogen as the electron donor, or (2) as electron carrier from the pyruvate phosphoroclastic system toward the sulfite-reducing system, using pyruvate as electron donor.l I J.-H. K i m a n d J. M. Akagi, J. Bacteriol. 163, 472 (1985).
METHODS IN ENZYMOLOGY,VOL. 243
Copyright © 1994by AcademicPress, Inc. All rights of reproduction in any form reserved.
[ 13]
FLAVODOXINS
189
5' C,H,OPO,H, I 4' CHOH I 3' CHOH
I
2' CHOH
I
1' OH, I -N
H,C
,,.,,"~6
H.C-
~
5~
0~
N
O
4 , ~ HN
"N"
0 FIO. 1. Structure of riboflavin Y-phosphate (FMN).
In the p r e s e n c e of iron in the culture medium of sulfate-reducing bacteria, h o w e v e r , the biosynthesis of flavodoxins is suppressed at the expense of ferredoxin. 2'3 F l a v o d o x i n s s e e m to be able to take o v e r the role of ferredoxins as electron d o n o r in the sulfite-reducing system. Purification T h e purification of D e s u l f o o i b r i o flavodoxins can be done according to the p r o c e d u r e s of L e Gall and Hatchikian, 4 M o u r a et al.,5 and Ifie et al. 6 Reagents and Materials
D E A E - 5 2 cellulose S e p h a d e x G-50 Tris-HC1 buffer (50 raM), p H 7.6 All steps are carried out at 4 ° unless specified otherwise. The D e s u l f o o i b r i o cells are suspended in 50 m M Tfis-HCl buffer (about 1 g of cells/ml buffer). The cells are subsequently disrupted by a French press and centrifuged at 30,000 g for 30 min. T h e supernatant is passed o v e r a DEAE-52 cellulose 2 H. D. Peck, Jr., and J. LeGall, Philos. Trans. R. Soc. London B. 298, 443 (1982). 3 S. G. Mayhew and M. L. Ludwig, Enzyme 12, 57 (1975). 4 j. LeGall and E. C. Hatchikian, C.R. Acad. Sci. Paris 264, 2580 0967). 5 I. Moura, J. J. G. Moura, M. Brusch, and J. LeGall, Biochim. Biophys. Acta 591, 1 0980). 6 K. Irie, K. Kobayashi, M. Kobayashi, and M. Ishimoto, J. Biochem. (Tokyo) 73, 353 (1973).
190
DISSIMILATORY SULFATE REDUCTION
[13]
column, equilibrated with 50 mM Tris-HCl buffer. The bound flavodoxin is eluted from the column with 0.15 M Tris-HC1 buffer, 0.4 M KC1, pH 7.6. This fraction is desalted over a Sephadex G-50 column (in 50 mM Tris-HCl, pH 7.6) or alternatively dialyzed against 50 mM Tris-HCl buffer, pH 7.6. The protein is then brought onto a DEAE-52 cellulose column (volume > 150 ml) equilibrated with Tris-HC1 buffer (50 mM, pH 7.6) and the flavodoxin is eluted from the column by a linear gradient of Tris-HCl (50 mM to 0.5 M). The flavodoxin fraction elutes at 0.30-0.35 M TrisHCI. As the flavodoxin fraction can still contain other protein components (mainly rubredoxin) this fraction is dialyzed against 50 mM Tris-HCl buffer (pH 7.6) and brought onto a second DEAE-52 cellulose column, which is then eluted with a linear gradient of Tris-HCl (0.25 to 0.40 M, pH 7.6). Redox Potentials Flavodoxins can occur in three redox states (Fig. 2). Of particular physiological importance3 is the transition between the one electron-reR
I
H3cWN'Ny°
OXIDIZED
N"
H+,e.llO2 0 RI FI3C~N~N~O SEMIQUINONE
eltO'
o
R I
HaC~NvN.,,~O HYDROQUINONE
0 FIG. 2. The structures of oxidized (quinone), one electron-reduced (semiquinone), and two electron-reduced (hydroquinone) flavin.
[13]
FLAVODOXINS
191
TABLE I REDOX POTENTIALSOF FLAVODOXINS(IN MILLIVOLTS)a
Desulfovibrio vulgaris CIostridium beijerinckii MP Megasphaera elsdenii Anabaena variabilis Anacystis nidulans Azotobacter vinelandii Azotobacter chroococcum Escherichia coli Klebsiella pneumoniae Riboflavin 5'-phosphate (FMN)
Ez
El
Ref.
- 143 - 92 - 115 - 195 - 221 - 165 - 115 -240 - 158 -314
- 435 - 399 - 372 - 390 - 447 -458 - 520 -410 -412 - 124
7 8 9 10 11 12 13 14 13 15
a E1 , the transition from semiquinone to the hydroquinone form; E 2, the transition from the oxidized to the semiquinone form. All values indicated are at pH 7.
duced state and the two electron-reduced state. The redox potentials of this t r a n s i t i o n a r e a m o n g t h e l o w e s t o b s e r v e d in n a t u r e , r a n g i n g f r o m - 3 0 5 to - 5 2 0 m V ( T a b l e I). 7-15 R e d u c t i o n o f o x i d i z e d f l a v o d o x i n to t h e s e m i q u i n o n e f o r m c a n e a s i l y be achieved by the addition of (chemical) reducing agents. However, o w i n g to t h e l o w r e d o x p o t e n t i a l o f t h e s e m i q u i n o n e - h y d r o q u i n o n e t r a n s i t i o n ( h i s t o r i c a l l y c a l l e d E 0 c o m p l e t e r e d u c t i o n o f D e s u l f o v i b r i o vulgaris f l a v o d o x i n is difficult to a c h i e v e a n d d e p e n d s o n t h e r e l a t i v e r e d o x p o t e n tials o f t h e e l e c t r o n a c c e p t o r ( f l a v o d o x i n ) a n d e l e c t r o n d o n o r . T h e m o s t w i d e l y u s e d e l e c t r o n d o n o r is d i t h i o n i t e . A t l o w c o n c e n t r a tions of flavodoxin (<0.5 mM) reduction can be accomplished rather easily by the near stoichiometric addition of a freshly prepared dithionite solution in p h o s p h a t e b u f f e r , p H 7 ( 1 0 0 - 2 0 0 m g / m l ) . I t is a d v i s a b l e to flush t h e buffer with (oxygen-free) argon before addition of dithionite. However, 7 G. P. Curley, M. C. Carr, P. A. O'Farell, S. G. Mayhew, and G. Voordouw, in "Flavins and Flavoproteins 1990" (B. Curti, S. Ronchi, and G. Zanetti, eds.), p. 429. de Gruyter, Berlin, 1991. 8 S. G. Mayhew, Biochim. Biophys. Acta 235, 276 (1971). 9 S. G. Mayhew, G. P. Foust, and V. Massey, J. Biol. Chem. 244, 803 (1969). l0 M. F. Fillat, G. Sandmann, and C. Gomez-Moreno, Bioehem. Biophys. Res. Commun. 1040, 301 (1990). 11 B. Entsch and R. M. Smillie, Arch. Biochem. Biophys. 151, 378 (1972). 12M. F. Taylor, W. H. Boylan, and D. E. Edmondson, Biochemistry 29, 6911 (1990). 13j. Deistung and R. N. F. Thorneley, Biochem. J. 239, 69 (1986). 14 H. Vetter, Jr., and J. Knappe, Hoppe-Seyer's Z. Physiol. Chem. 352, 433 (1971). 15R. F. Anderson, Biochim. Biophys. Acta 772, 158 (1983).
192
DISSIMILATORY SULFATE REDUCTION
[13]
at high flavodoxin concentrations (>0,5 mM) reduction to a 100% hydroquinone form is virtually impossible to achieve at pH values below 7,5 and at ionic strengths below 100 mM. J6At high concentrations of flavodoxins (> 1 mM) complete reduction to the hydroquinone form by dithionite (at 200-mg/ml stock solution) can be achieved when keeping the pH of the sample above pH 8. (The flavodoxin sample should be oxygen free, as reduction of the residual oxygen by dithionite gives rise to pH changes that make reduction more difficult.) The contamination of sodium dithionite with small amounts of (bi)sulfite may cause oxidation of flavodoxin. 16 Therefore it is essential to use the highest purity of dithionite available. Reduction of flavodoxins can also be accomplished by illumination (50- to 100-W tungsten lamp) of an anaerobic solution of flavodoxin in the presence of ethylenediaminetetraacetic acid (EDTA) (50 mM) and of a catalytic amount of 5-deazariboflavin. The reduction to the semiquinone form is completed typically within I hr. Continued illumination (several hours) yields the hydroquinone. A major advantage over reduction by dithionite is that by using EDTA and light no spectral disturbance occurs in the region of 280-380 nm.17 The flavodoxin redox potentials can be determined by controlled titration with dithionite and subsequent measurement of the concentration of the semiquinone radical by electron paramagnetic resonance (EPR). 1a,19 Typically a deaerated and argon-flushed buffered solution of 20 to 50/xM flavodoxin and a l0/xM series of mediator dyes is titrated with dithionite in buffer under a constant flow of argon. The potential of the solution is measured between a platinum electrode and a calomel or Ag/AgC1 reference electrode. After each addition of dithionite, and after allowing the solution to equilibrate, a sample is anaerobically injected into an EPR tube and frozen in liquid nitrogen. The potential range of the mediators must be sufficient to buffer the potential at any point of the titration. The use of mediators accepting two electrons is recommended to avoid interference of mediator radical signals with the protein semiquinone signal. However, for the titration of E~, no two-electron mediators are available. Mediators accepting one electron can be used if the concentrations of these mediators are low with respect to the protein and the sharp mediator radical signal can be subtracted from the broader protein signal. The relative concentration of semiquinone as determined from the EPR spectra (corrected for dilution) is plotted versus the potential and fitted to the Nernst equation to determine the redox potentials. 16 S. G. Mayhew, Eur. J. Biochem. 85, 535 (1978). 17 V. Massey and P. Hemmerich, Biochemistry 17, 9 (1978). 18 G. S. Wilson, this series, Vol. 54, p. 396. 19 p. L. Dutton, this series, Vol. 54, p. 411.
[13]
FLAVODOXINS
193
Alternatively, a potentiometric titration can be performed by measuring the concentrations of quinone, semiquinone, and hydroquinone spectrophotometricaUy. The reduction is performed by adding dithionite (E2), by hydrogen in the presence of hydrogenase (E0, or by photoreduction (E2 and E0. The potential of the solution can be calculated from the amount of reductant added (or partial hydrogen pressure) or from the redox state of a reference mediator dye, or may be directly measured as described. 17-zl The quinone-semiquinone potential can also be determined by the spectrophotometric xanthine/xanthine oxidase titration described by Mass e y . 22 The method uses the low potential of the xanthine/ureate couple (-350 mV at pH 7) together with the ability of xanthine oxidase and flavoproteins to exchange electrons with redox dyes. Neither xanthine nor ureate interfere in the near ultraviolet (UV)-visible range. Typically, a buffered solution of 10 to 20 ~M flavodoxin, 200-300/xM xanthine, a redox dye with a potential within 30 mV from the unknown, and 2/zM methyl or benzyl viologen is placed in an anaerobic cuvette, deaerated, and flushed with argon. It is preferable to use a redox dye with a measurable difference in absorption between the oxidized and reduced form at an isosbestic point of the flavodoxin. At t = 0 an anaerobic solution of xanthine oxidase is added and spectra are recorded. The amount of xanthine oxidase must be low (10-50 nM) to ensure equilibrium conditions. The complete reduction is typically completed within 1 to 2 hr and about 50-100 spectra are recorded. The potential of the flavodoxin can be determined by plotting its log(ox/red) value versus the log(ox/red) value of the redox dye. The slope of the plot is unity when the two couples involve the same number of electrons. The difference between the potentials can be calculated (using the Nernst equation) from the log(ox/red) value of the flavodoxin at the point where the log(ox/red) value of the dye is zero. As an alternative for the titrative determination of redox potentials, direct electrochemistry of redox proteins has become feasible over the last 10 years.Z3-31Direct electrochemistry of flavodoxins has been reported 20 M. Dubourdieu, J. LeGall, and V. Favaudon, Biochim. Biophys. Acta 376, 519 (1975). 21 S. G. Mayhew and V. Massey, Biochim. Biophys. Acta 315, 181 (1973). 22 V. Massey, "Proceedings of the Tenth International Symposium on Flavins and Flavoproteins," p. 59. de Gruyter, Berlin, 1991. 23 C. Van Dijk, J. W. Van Leeuwen, and C. Veeger, Bioelectrochem. Bioenerg. 9, 743 (1982). 24 W. R. Hagen, Eur. J. Biochem. 182, 523 (1989). 25 L. H. Guo, H. A. O. Hill, G. A. Lawrence, G. S. Sanghera, and D. J. Hopper, J. Electroanal. Chem. 266, 379 (1989). 26 p. Bianco, J. Haladjian, A. Manjaoui, and M. Bruschi, Electrochim. Acta 33, 745 (1988). 27 F. A. Armstrong, J. N. Butt, and A. Sucheta, this series, Vol. 227, p. 479. 28 F. A. Armstrong, H. A. O. Hill, B. N. Oliver, and N. J. Walton, J. Am. Chem. Soc. 106, 921 (1984).
194
DISSIMILATORY SULFATE REDUCTION
[13]
by Van Dijk, 23 Armstrong, Hill, and co-workers. 27,28 Van Dijk, 23 using differential and normal pulse polarography, determined the quinone/semiquinone ( - 114 mV, pH 7.4) and semiquinone/hydroquinone ( - 392 mV, pH 7.4) couples of M. elsdenii flavodoxin adsorped at the mercury electrode. At the pyrolytic graphite electrode, Armstrong et al. 2s only found the semiquinone/hydroquinone couple of M. elsdenii flavodoxin (-318 mV, pH 5.0) using square wave voltammetry and in the presence of MgCI/ and Cr(NH3)63+. Bianco et al. 26 determined the semiquinone/hydroquinone couple of D. vulgaris (Hildenborough) flavodoxin (-430 mV, pH 7.6) at the pyrolytic graphite electrode using differential pulse voltammetry and cyclic voltammetry. Cyclic voltammetry of the flavodoxins isolated from Azotobacter choococcum at a polished edge-plane graphite electrode has been performed29 in the presence of the cationic aminoglycoside neomycin. The semiquinone-hydroquinone potentials of two distinct flavodoxins were found (-305 and -520 mV at pH 7.4). Our group has investigated the electrochemistry of D. vulgaris Hildenborough. 3°,3~ In the classic three-electrode setup and semiinfinite diffusion, a bulk solution of about 1 mEand a protein concentration of 100/zM is required. In the setup developed in our laboratory the volume of the bulk is reduced to less than 20/xl with full retention of semiinfinite diffusion down to a scan rate of 1 mV/sec. ~4 The working electrode is a dismountable inverted disk of glassy carbon (15-ram diameter and 2-mm height, type V25; obtained from Le Carbon Loraine, Paris). Prior to each electrochemical measurement the disk is polished firmly with polishing cloth with 6-/~m diamond lapping compound (Engis, Ltd., Kent, England), rinsed thoroughly with water, and dried. To activate the electrode it is exposed for 30 sec to a methane flame from a Bunsen burner. The polished working surface is never in direct contact with the flame, and if any inhomogeneity is detected on the surface the polishing and glowing are repeated. The counterelectrode is a microplatinum electrode (P-1312; Radiometer) and the reference electrode is a saturated calomel electrode (K-401; Radiometer). After mounting the electrodes the cell is flushed with wet argon. A deaerated, argon-flushed sample is then transferred with a gas-tight Hamilton syringe to the tip of the reference electrode and the working electrode holder is pushed up until the shape of the droplet is approximately cylindrical. The electrodes are connected to an Autolab 10 potentiostat (Eco Chemie) controlled by GPES software (version 2.0) (Eco Chemie) on a PC. 29 S. Bagby, P. D. Barker, H. A. O. Hill, G. S. Sanghera, B. Dunbar, G. A. Ashby, R. R. Eady, and R. N. F. Thorneley, Biochem. J. 277, 313 (1991). 30 H. A. Heering and W. R. Hagen, J. Inorg. Biochem. 51, 25 (1993). 31 H. A. Heering and W. R. Hagen, unpublished results (1994).
[ 13]
FLAVODOXINS
-66o
'
-4'o0
-2bo
195
b
'
260
Em (mY)
FIG. 3. Staircase cyclic voltammogram (20 mV/sec) of 6 /zl of 0.14 mM D. oulgaris flavodoxin in 20 mM potassium phosphate, pH 7.0: before and after addition of 0.5 pJ of 50 mM neomycin.
Staircase cyclic voltammetry (SCV) was done with steps of 2.44 inV. Differential pulse polarography (DPP) was done in both directions (after a 60-sec equilibration at the starting potential) with steps of 2.44 mV and 250 msec, modulated by pulses of 20 mV and 100 msec (Fig. 3). The experiments were performed at a temperature of 22 - 1° and the potentials have been recalculated with respect to the normal hydrogen electrode (NHE), using the potential of - 2 4 6 mV (NHE) of the saturated calomel electrode (SCE). The promotor neomycin B (Sigma, St. Louis, MO) is added from a 50 mM solution, titrated to pH 7.0 with NaOH. The pH dependence is measured with SCV and DPP. The SCV measurements are performed on droplets of 5 ~1 of 0.11 mM flavodoxin and 3.3 mM neomycin in 20 mM citrate, 20 mM bistrispropane, and 20 mM CAPS, titrated with HCI or NaOH and adjusted to ionic strength/z = 0.2 with NaC1. The DPP measurements were performed on droplets of 5 /xl 0.13 mM flavodoxin and 3 mM neomycin in the same buffer. In all experiments the fully oxidized flavodoxin (as determined spectroscopically) was used. As in the experiments of Armstrong e t a / . 27'28 and of Bagby et a1.,29 the first reduction (quinone to semiquinone) is not detectable at the electrode and the second reduction step is detected only after addition of a cationic promotor such as neomycin. The cations are believed to form a
196
DISSIMILATORY SULFATE REDUCTION
[13]
O. Xin: p K a = 4 . 8
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bridge between the negative charges of both the electrode and flavodoxin (pl = 3.6). Without neomycin (at pH 7.0) only one redox couple is found at a potential of -217 mV (NHE) with a peak current proportional to the scan rate. This response is caused by FMN, dissociated from the protein and adsorbed onto the electrode. The potential and the PKa of 6.6 are equal to those measured for free FMN in solution. After addition of neomycin one additional response is observed with a peak current proportional to the square root of the scan rate and the characteristics of a oneelectron transition. The potential of -409 mV (NHE) is near the chemically determined potential of the semiquinone-to-hydroquinone reduction. 2°'32 The observed PKa of 4.8 is, however, much lower than the reported value of 6.620 to 6.8. 32 This difference between the pK a at the electrode and the one reported for the chemically reduced flavodoxin might be caused by a conformational change near the electrode. The measured pKa of 4.8 can be due to the altered environment of the amino acid residue responsible for the redox-linked PKa. Alternatively, the residue might be blocked or no longer in the vicinity of the isoalloxazine. The measured pK a of 4.8 can be due to protonation of the isoalloxazine at N-1. See Fig. 4. The midpoint potential of the quinone to semiquinone reduction (as determined by EPR titration with dithionite) is -113 mV (NHE) at pH 7.0. This value is in agreement with reported potentials. 2°'32 The absence of the quinone to semiquinone couple in the voltammograms can be ex32 G. P. Curley, M. C. Carr, S. G. Mayhew, and G. Voordouw, Fur. J. Biochem. 202, 1091 (1991).
[ 13]
FLAVODOXINS
197
plained 3°'31 by the known fast comproportionation mechanism of one fully reduced and one fully oxidized flavodoxin to two semiquinone flavodoxin molecules 2° in combination with a very slow (virtually not occurring) reduction of the oxidized to the semiquinone form. 21 By consequence, only a few molecules (catalytic amount) of semiquinone are sufficient to generate equilibrium concentrations of semiquinone and hydroquinone near the electrode surface and the development of the voltammogram without a visible first redox couple. A catalytic amount of free FMN may also act as a mediator to generate the first molecules of semiquinone, Amino Acid Sequences and Structure The amino acid sequences of several flavodoxins are known (Table II). The homology between these sequences ranges from 20 to 40%. As alignment solely on the basis of sequences is known to be difficult, structural alignment was investigated using the structures of the flavodoxins from Chondrus crispus, D. vulgaris, and Clostridium beijerinckii. The structurally conserved regions were then determined. On the basis of these conserved regions, the alignment of the other flavodoxin sequences was performed using the Clustal V alignment program. 33 Conserved residues are marked in bold in Table I I . 34-46 As is evident from this alignment only five residues are totally conserved. The first four of these residues are embedded close to each other in a hydrophobic pocket in the protein. These four residues may either be of importance for electron transfer processes or may be important in the folding process of flavodoxins. As can be inferred from the amino acid sequences, flavodoxins are acidic proteins. Their net negative charges ranges from - 1 0 to -20, and their isoelectric points are situated around pH 4. The low isoelectric points, high 33 D. G. Higgins and P. M. Sharp, Gene 73, 237 (1988). 34 M. Dubourdieu and J. L. Fox, J. Biol. Chem. 252, 1453 (1977). 35 L. R. Helms and R. P. Swenson, Biochim. Biophys. Acta 1089, 417 (1991). 36 L. R. Helms, G. D. Krey, and R. P. Swenson, Biochem. Biophys. Res. Commun. 168, 809 (1990). 37 M. Tanaka, M. Haniu, K. T. Yasunobu, and S. G. Mayhew, J. Biol. Chem. 249, 4393 (1974). 38 D. Santangelo, D. T. Jones, and D. R. Woods, J. Bacteriol. 173, 1088 (1991). 39 M. Tanaka, M. Haniu, K. T. Yasunobu, S. G. Mayhew, and V. Massey, J. Biol. Chem. 249, 4397 (1974). 4o S. Wakabayashi, T. Kimura, K. Fukuyama, H. Matsubara, and L. J. Rogers, Biochem. J. 263, 981 (1989). 41 K. G. Leonhardt and N. A. Straus, Nucleic Acids Res. 17, 4384 (1989). 42 D. E. Laudebach, M. E. Reith, and N. A. Straus, J. Bacteriol. 170, 258 (1988). 43 L. T. Bennett, M. R. Jacobson, and D. R. Dean, J. Biol. Chem. 263, 1364 (1988). 44 C. Osborne, L. M. Chen, and R. G. Matthews, J. Bacteriol. 173, 1729 (1991). 45 W. Arnold, A. Rump, W. Klipp, U. B. Priefer, and A. J. Puehler, J. Mol. Biol. 203, 715 (1988). 46 y . Jouanneau, P. Richard, and C. Grabau, Nucleic Acids Res. 18, 5284 (1990).
198
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stability, and high charge density make it possible to prepare flavodoxin solutions of up to 400 mg/ml. At these high concentrations no aggregation phenomena can be observed as is evident from ~H nuclear magnetic resonance (NMR) studies at these concentrations. 47'48 This property, in combination with their low molecular weight, makes flavodoxins good candidates for structure determination using multidimensional NMR techniques. From such multidimensional NMR studies it became clear that the solution and crystal structures of D. vulgaris flavodoxin are virtually identical. 48 The tertiary structures of all flavodoxins share a common polypeptide fold consisting of a central parallel fl sheet with five strands surrounded on both sites by helices. The FMN is strongly bound via H bonds on one side of the protein with the isoalloxazine moiety exposed to the solvent. The loop regions that surround the isoalloxazine ring (residues 59-64 and 94-104 in D. vulgaris flavodoxin) have hardly any conserved sequences. Nevertheless, in general the isoalloxazine ring seems to be sandwiched between two aromatic residues (W60 and Y98 in D. vulgaris flavodoxin). In some flavodoxins one of the two aromatic residues is replaced by a hydrophobic residue (M, L, or I). It appears, on comparison of the known structures and amino acid sequences (Table II), that the isoalloxazine ring needs to be shielded from the solvent by these residues for the establishment of the low redox potentials. 49 The reader is referred to detailed reviews on the three-dimensional structure characteristics of flavodoxins.50
Apoflavodoxin Preparation Apoflavodoxin can be prepared according to the following procedure (this procedure contains slight modifications of the original procedure of Wassink and Mayhew 51).
Procedure 1. Cold trichloroacetic acid (TCA)(T = 4°) at a concentration of 40% (w/v) is slowly added to a cold flavodoxin solution (concentration 0.2-0.4 mM, 4°) in 0.1 M phosphate buffer (pH 7-8)-0.3 mM EDTA, to give a final concentration of 5% TCA. The flavodoxin solution should preferably be shielded from light. After the addition of TCA the solution 47 S. S. Wijmenga and C. P. M. van Mierlo, Eur. J. Biochem. 195, 807 (1991). 48 S. Peelen, J. Vervoort, and J. LeGall, unpublished results (1994). 49 j. Vervoort, Curt. Opin. Struct. Biol. 1, 889 (1991). 5o M. L. Ludwig and C. L. Luschinsky, in "Chemistry and Biochemistry ofFlavoenzymes" (F. MOiler, ed.), Vol. 3, p. 427. CRC Press, Boca Raton, Florida, 1991. 5z j. H. Wassink and S. G. Mayhew, Anal. Biochem. 68, 609 (1975).
[13]
FLAVODOX|NS
201
is centrifuged at 10,000 g for 10 min. The white precipitate can be dissolved in 0.1 M potassium phosphate-0.3 mM EDTA (pH 7-8). As the procedure does not give 100% apoprotein in one run, it is advisable, when necessary, to repeat the procedure. The apoprotein prepared in this way is stable for a long period: more than 1 month at 4° up to 1 week at room temperature. Procedure 2. A milder procedure for apoflavodoxin preparation is as follows: Dialyze 0.2-0.4 mM flavodoxin in 0.1 M potas sium phosphate-0.3 mM EDTA (pH 7-8) four times against 250 ml of 2 M KBr in 0.1 M sodium acetate buffer, pH 3.9, with 0.3 mM EDTA (total time, 48 hr). The precipitate can be redissolved in 0.1 M potassium phosphate (pH 7.0)-0.3 mM EDTA. The disadvantage of this procedure is the long time span needed to prepare the apoprotein. The apopreparation procedure does not lead to irreversible modifications of the apoprotein. 31p and 1H NMR studies of flavodoxins from D. vulgaris and also from M. elsdenii, before (native) and after apopreparation followed by reconstitution with FMN, give identical spectra. From this the firm conclusion can be drawn that the recombined protein (apoprotein recombined with FMN) and the native protein have the same conformation. Reconstitution of Holoflavodoxin from Apoprotein and Ribofavin 5'-Phosphate A wealth of information is available about the reconstitution of holoflavodoxin from its constituents, the apoprotein and prosthetic group. For details about the thermodynamics and kinetics of flavin binding the reader is referred to the review of Mayhew and Ludwig) Apoflavodoxins from Clostridium species and M. elsdenii are specific for binding of flavins at the FMN level) 2,53 The apoflavodoxins from Azotobacter vinelandii and D. vulgaris form tight complexes not only with FMN but also with riboflavin and lumiflavin analogs. 54,55 Apoflavodoxins tightly bind FMN at neutral pH. For both M. elsdenii and D. vulgaris flavodoxin, the dissociation constant (Kd) of the complex is in the range of 10-10 M.52,56 Stabilization of the interaction between apoprotein and prosthetic group is de52 S. G. Mayhew, Biochim. Biophys. Acta 235, 289 (1971). 53 j. A. D'Anna and G. Tollin, Biochemistry 11, 1073 (1972). 54 D. E. Edmondson and G. Tollin, Biochemistry 10, 113 (1971). 55 D. E. Edmondson and G. Tollin, Biochemistry 10, 133 (1971). 56 j. Vervoort, W. J. H. van Berkel, S. G. Mayhew, F. Mt~ller, A. Bacher, P. Nielsen, and J. Legall, Eur. J. Biochem. 161, 749 (1986).
202
DISSIMILATORY SULFATE REDUCTION
[13]
creased at pH values below 5 and is also dependent on the ionic strength and type of anions present in solution. 57 Binding of FMN at neutral pH is rapid and results in almost complete quenching of flavin fluorescence?2 This property has been used with the apoflavodoxin from M. elsdenii to assay for FMN and FAD in mixtures and to analyze commercial FMN preparations for their content. 51 Commercial FMN preparations contain 25-30% of fluorescent impurities. 51,58 Synthesis of FMN by chemical phosphorylation of riboflavin yields 4'-FMN as a main contaminant. 59 By 31p NMR it was shown that commercial FMN also contains considerable amounts of the 3' and 2' isomers and other phosphorus-containing compounds. 6° The impurities (including riboflavin) and 5'-FMN are conveniently separated by reversedphase high-performance liquid chromatography (HPLC) 61and preparative amounts of each compound can be obtained in pure form. 62'63On the basis of various chemical properties and enzymatic hydrolysis, the structures of the phosphorylated impurities have been assigned t o 61'62 riboflavin 4'-phosphate (4'-FMN), riboflavin 3'-phosphate (Y-FMN), riboflavin 2'-phosphate (2'-FMN), riboflavin 4',5'-bisphosphate (4',5'-FBP), riboflavin 3',5'-bisphosphate (3 ',5'-FBP), riboflavin Y,4'-bisphosphate (3',4'FBP), riboflavin 2',5'-bisphosphate (2',5'-FBP), riboflavin 2',4'-bisphosphate (2' ,4'-FBP) and riboflavin 4',5'-cyclophosphate (4',5'-FCP), respectively. The occurrence of these flavin isomers is explained by the acidcatalyzed migration of the phosphoric acid groups in both the riboflavin mono- and bisphosphates. 59'62 At neutral pH, the isomerization reactions are slow as compared to hydrolysis of the phosphoester bond. 6z For a long time it was thought that apoflavodoxin is highly specific for 5'-FMN. 5~Separation of the isomeric riboflavin mono- and bisphosphates, however, revealed that apoflavodoxin from M. elsdenii also binds 3',5'FBP tightly. 6~This compound makes up 2% of the total amount of commercial FMN. 62Megasphaera elsdenii apoflavodoxin reconstituted with Y,5'FBP is fully active as an electron carrier by transferring reduction equivalents from H2 via hydrogenase to metronidazole.6~ This suggested that the additional phosphate group does not influence the properties of the complex. A more thorough kinetic, thermodynamic, and spectral analysis of the complexes of 3',5'-FBP and the apoflavodoxins from M. elsdenii 57 R. Gast, B. E. Valk, F. Mfiller, S. G. Mayhew, and C. Veeger, Biochim. Biophys. Acta 446, 463 (1976). 58 V. Massey and B. E. P. Swoboda, Biochem. Z. 339, 474 (1963). 59 G. Scola-Nagelschneider and P. Hemmerich, Eur. J, Biochem. 66, 567 (1976). 6o C. T. W. Moonen and F. MOller, Biochemistry 21, 408 (1982). 61 p. Nielsen, P. Rauschenbach, and A. Bacher, Anal. Biochem. 130, 359 (1983). 62 p. Nielsen, J. Harksen, and A. Bacher, Eur. J. Biochem. 152, 465 (1985). 63 p. Nielsen, P. Rauschenbacher, and A. Bacher, this series, Vol. 122, p. 209.
[14]
RUBREDOXIN IN CRYSTALLINE STATE
203
and D. vulgaris confirmed this idea. 56Both apoflavodoxins bind 3',5'-FBP somewhat more weakly than 5'-FMN owing to slower association rate constants. The redox potentials of the artificial complex, however, are similar to the values reported for the native protein. 31p NMR experiments revealed that the 3'-phosphate group is accessible for Mn 2÷ and indicates that this group is located close to the protein surface. 56 This is in sharp contrast with the 5'-phosphate group, which is dianionic 6° and buried in both proteins. The 5'-phosphate group therefore determines the specificity of flavin binding, whereas introduction of the 3'-phosphate group only slightly influences the protein conformation. The latter conclusion is in full accordance with results obtained from the crystal structure of D. vulgaris flavodoxin as well as ~H NMR results, which show that the 3'-OH of the ribityl side chain is oriented toward bulk solvent. 48,64 64 W. Watt, A. Tulinsky, R. P. Swenson, and K. D. Wautenpaugh, J. Mol. Biol. 218, 195 (1991).
[14] R u b r e d o x i n in C r y s t a l l i n e S t a t e By
LARRY C . SIEKER, RONALD
E.
STENKAMP,
and
JEAN L E G A L L
General Remarks Rubredoxin (Rd) is one of the simplest of iron proteins and has been found, thus far, only in certain microorganisms. 1 The initial report and characterization of a rubredoxin was done by Lovenberg and Sobel in 1965. 2 Rubredoxins are composed of 45 to 54 amino acid residues with molecular weights ranging from 5000 to 6000 and contain 1 iron atom liganded by 4 cysteine residues. The iron center can be reversibly reduced at a redox potential near 0 inV.1 Although many rubredoxins have been detected and isolated from a variety of bacteria, only 13 of the rubredoxins have had amino acid sequences determined. Figure 1 shows the amino acid sequence alignment of the 13 rubredoxins. Because this chapter is primarily directed to the sulfate-reducing bacteria we have chosen to divide these rubredoxins into three categories. Figure la lists the Rds from the sulfate-reducing Desulfooibrio species, Fig. lb shows the Rds from a mixed assortment of bacteria, and Fig. lc contains the thermophilic Rds. i T. G . Spiro, "Iron-Sulfur Proteins." Wiley (Interscience), New York, 1982. 2 W. Lovenberg and B. E. Sobel, Proc. Natl. Acad. Sci. U.S.A. 54, 193 (1965).
METHODS IN ENZYMOLOGY,VOL. 243
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