Dimerization of Azotobacter vinelandii flavodoxin (azotoflavin)

ARCHIVES

OF

BIOCHEMISTRY

AND

Dimerization

BIOPHYSICS

170,

326-333

of Azotobacter vine/an&i (Azotoflavin) DUANE

Department

(1975)

of Cell Physiology,

C. YOCH

University Received

Flavodoxin

of California, March

Berkeley,

California

94720

25, 1975

Molecular weight determinations by gel filtration of Azotobacter vinelandii flavodoxin (azotoflavin) which had been stored aerobically for various periods of time indicated that this flavoprotein electron carrier could exist in the monomeric (M, = 23,000) or dimeric CM, = 46,000) form. This conclusion was substantiated by sedimentation velocity centrifugation analysis and sodium dodecyl sulfate electrophoresis data. The absorbance ratios (A2,4:A,,) of both monomer and dimer were identical, indicating the same ratio of flavin to protein in both species and that dimerization was a simple aggregation of two flavoprotein molecules. Because the flavodoxin dimer could be converted to predominantly the monomeric form by 2-mercaptoethanol, this was taken as preliminary evidence that dimerization is the result of a disulfide bridge between the cysteinyl group of two subunits and that 2-mercaptoethanol breaks this disulfide bond by reducing it back to its original cysteinyl sulfhydryl form. The oxidized form of monomeric flavodoxin was reduced by sodium dithionite to the semiquinone form at rates two to four times faster than was the oxidized form of the dimer. Similarly, the semiquinone form of the monomer was reduced to hydroquinone at rates somewhat faster than the comparable reaction of the dimer. The semiquinone forms of both monomer and dimer were oxidized by oxygen at approximately the same rate.Only monomeric flavodoxin had biological activity-it coupled the reducing power of illuminated chloroplasts to Azotobacter nitrogenase. The dimer was completely inactive in this reaction unless it was supplemented with about 10e6 M methyl viologen (which by itself was not active). The biological activity of the monomer (and its absence in the dimer), along with the probable ratios of monomer to dimer in Go, and the possible significance of this ratio are discussed.

A flavoprotein from Azotobacter vinelandii whose semiquinone form is unusually stable to both reduction and air oxidation has been isolated independently in three different laboratories. This protein was first discovered by Shethna et al. (1,2) who found it during the isolation of iron-sulfur proteins from Azotobacter and reported it as a previously unidentified flavoprotein of unusual properties. It has been commonly called the “Shethna flavoprotein.” Bulen et al. (3) reported a low molecular weight flavoprotein that separated during purification of the nitrogenase complex of A. vinelandii; it was subsequently crystallized and further characterized by Hinkson and Bulen (4) who called it the “free-radi-

cal flavoprotein” from Azotobacter. Finally, in 1969, Benemann et al. (5) showed that an electron carrier preparation from A. vinelandii was capable of coupling the reducing power of illuminated spinach chloroplasts to the nitrogenase of this organism. Purification of the active component led to the isolation of two ferredoxins (6,7) and a flavoprotein that was called “azotoflavin”. The characteristics described for these three flavoproteins (1, 2, 4, 5) indicated that they were the same protein. Benemann et al. (5) believed that this flavoprotein differed significantly enough from the class of flavoproteins known as flavodoxins to warrant a new name. It has recently been suggested that flavodoxins 326

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0 1975 by Academic Press, of reproduction in any form

IPC. reserved.

DIMERIZATION

OF AZOTOBACTER

be redefined simply as FMN-containing, electron-transferring proteins and that the flavoprotein from A. vinelandii be included in this category (8-10). This suggestion is supported by similarities in the Nterminal region of the amino acid sequence of the Azotobacter flavoprotein and other flavodoxins (11). The original definition of a flavodoxin as a flavoprotein which substitutes for ferredoxin in cells which are starved for iron (12) has proven far too restrictive, as a number of bacteria are now known to produce both flavodoxin and ferredoxin under conditions of normal iron metabolism. Furthermore, variations in biological activity, molecular weight, and oxidation-reduction characteristics indicate that flavodoxins are a more heterogeneous group of proteins than was originally thought (12) and that azotoflavin, although it differs in some characteristics from the clostridial flavodoxins, belongs in the broad group of flavoprotein electron carriers known as flavodoxins. It has been observed in this laboratory that nearly homogeneous preparations of Azotobacter flavodoxin that have been stored frozen under air for a number of months have several characteristics that are substantially different from those of the original material. First, while maintaining its spectral integrity, the flavoprotein lost much of its activity as an electron carrier in the nitrogenase reaction and, secondly, it eluted from Sephadex columns as two species with different molecular weights, one approximately twice the molecular weight of the other. The latter observation suggested dimerization of the flavoprotein. Hinkson (13) had previously observed that preparations of this flavoprotein migrated as two barely separated bands on electrophoresis. Because it migrated as a single band when reduced by 2mercaptoethanol [D. E. Edmondson, cited by Hinkson (13)], a possible monomer-dimer relationship was suggested (13). In this paper evidence is presented for dimerization of Azotobacter flavodoxin, the type of bonding that may be involved in this protein-protein interaction is suggested, and the biological activity and

327

FLAVODOXIN

some of the oxidation-reduction properties of the monomer and dimer are compared. METHODS

Purification

of Azotobacter (Azotoflavin)

Flavodoxin

The flavodoxin used in this study was isolated from Azotobacter uinelundii strain OP (obtained from J. R. Benemann) (see Note added in proof). The cells were grown under Nz-fixing conditions (5) and flavodoxin (azotoflavin) was isolated from a 40% acetone + 5% Triton X-100 solution and separated from the ferredoxin fraction as previously described by Yoch and Arnon (7). After dialysis against 20 rnru phosphate buffer, pH 7.4 (hereafter referred to as the buffer), various preparations of crude flavodoxin were stored (under air) at -20°C for periods of lo-18 mo. Flavodoxin was subsequently purified by chromatography on a DEAE-cellulose column (3 x 20 cm) which was equilibrated with buffer and eluted with a linear gradient of NaCl. The gradient was made from 200 ml of buffer containing 0.2 M NaCl and 200 ml of buffer containing 0.5 M NaCl. All fractions containing flavoprotein with an absorbance at 452 nm greater than 0.25 were pooled and concentrated on a small DEAE-cellulose column (2 x 5 cm) which was eluted with buffer containing 0.8 M NaCl. The concentrated flavoprotein was chromatographed on a 2.5 x 90-cm Sephadex G-100 (superfine grade) column from which it eluted as two major bands of about equal size. Fractions of both the high and low molecular weight species with an A2,4:A452 absorbance ratio of less than 4.9 were pooled and stored separately at -20°C under an atmosphere of argon. (These flavoprotein species were the flavodoxin dimer and monomer, respectively; see Results section.) Flavoprotein with an absorbance ratio of 4.7 was tentatively assumed to be pure because Hinkson and Bulen (4) had shown that redissolved crystals of this protein have an A214:A452 absorbance ratio of 4.7. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis subsequently showed that these flavoprotein preparations were homogeneous.

Biological

Activities

As a measure of flavodoxin activity, the monomeric and dimeric forms of the flavoprotein were tested for their ability to mediate electron transfer between illuminated spinach chloroplast fragments [prepared as described by Yoch (1411 and Azotobacter nitrogenase, as described by Benemann et al. (5). Nitrogenase activity was measured by the reduction of acetylene to ethylene [Schollhorn and Burris (15) and Dilworth (16)] as determined by gas chromatography [Hardy et al. (1711.

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Molecular

C. YOCH

a

Weight Determinations

The molecular weights of the Azotobucter flavodoxin monomer and dimer were determined by gel filtration on Sephadex G-100 (superfine grade) columns by the method of Andrews (18). The column was equilibrated with 20 mM phosphate buffer (pH 7.4) and eluted at a rate of approximately 8.0 ml/h. The following proteins (5 mg of each) of known molecular weight were used for calibration: cytochrome c, 12,400; sperm whale myoglobin, 17,800; chymotrypsinogen A, 25,000; and ovalbumin, 45,000. In addition, the molecular weight was determined by sedimentation velocity centrifugation at 20°C: with a Spinco model E analytical ultracentrifuge. The flavoprotein was analyzed at concentrations of l-4 mg of protein/ml in 20 mM phosphate buffer (pH 7.4). The diffusion coefficient of dimeric flavodoxin used in these calculations was determined from gel filtration as described by Andrews (18).

Electrophoresis Purity of flavoprotein species was monitored by disc gel electrophoretic techniques as described by Davis (19). Polyacrylamide gel electrophoresis was carried out at room temperature using 20% acrylamide and Tris-glycine buffer (pH 8.3). SDS-polyacrylamide gel electrophoresis was performed according to Weber and Osborn (201. Both SDS and non-SDS gels were stained with Coomassie brilliant blue as previously described (201; however, non-SDS gels required only about 30 min of staining, as opposed to 2-3 h for the SDS gels. RESULTS

Evidence

for Dimerization

On disc gel electrophoresis, Azotobacter vinelandii flavodoxin (azotoflavin) preparations with an A274:A452 absorbance ratio of 4.7 [suggesting near homogeneity (4)l always showed, along with the major flavoprotein band, a minor band. During the purification steps, this minor “contaminating” band was found only in those fractions that containedflavoprotein. Fresh preparations of flavodoxin showed only a faint band and the size of the band increased with the age of the preparation. This is exemplified in Fig. 1, which shows the electrophoresis pattern of a 2- to 3-wk-old flavodoxin preparation (Fig. la) compared to a preparation that was approximately 10 mo old (Fig. lb). The older flavodoxin preparation can also be divided into two yellow, distinctly

b

+ FIG. 1. Polyacrylamide gel electrophoresis of Azotobacter flavodoxin (a) 30 pg of protein several weeks after preparation and (b) 110 pg of protein 10 mo after preparation (stored at -20°C under air). Gels stained with Coomassie brilliant blue.

separated fractions by gel filtration on a Sephadex G-100 column (Fig. 2). Precalibration of this column with different protein markers (insert to Fig. 2) permitted an estimation of the molecular weight of the two fractions. The species with a higher molecular weight (Peak A) had a molecular weight of 46,000, whereas the low molecular weight flavoprotein species (Peak B) had a molecular weight of about 24,000. Because the molecular weight ofA. vinelandii flavodoxin was previously found to be 23,000 (211, these observations suggested that the 46,000 M, flavoprotein (Peak A) was dimeric Azotobacter flavodoxin. The molecular weights of the flavoproteins from peaks A and B (Fig. 2) were also determined by sedimentation velocity centrifugation. The sedimentation coefficient, S 20,W,of Peak A flavoprotein was found to be 3.5. Using a diffusion coefficient of 7.25 x lo-‘, determined by gel filtration techniques (Yoch, unpublished data) and the partial specific volume (0.72) reported by Edmondson and Tollin (21) for the Shethna flavoprotein (Azotobacter vinelandii flavodoxin), the molecular weight of Peak A flavoprotein was calculated to be approxi-

DIMERIZATION

OF AZOTOBACZ’ER

’ i

!

150 Effluenf

200

250

1mfJ

FIG. 2. Elution pattern showing a separation of monomeric and dimeric Azotobocter flavodoxin by gel filtration on Sephadex G-100 (superfine grade). Peak A, dimeric flavodoxin; Peak B, monomeric flavodoxin. Column dimensions: 2.5 x 80 cm. Column equilibrated with 20 mM phosphate buffer (pH 7.3) containing 500 mM NaCl. Insert: The Sephadex G-100 column had previously been calibrated with standard proteins so that molecular weights could be estimated (see Methods for details). Standard proteins: (1) cytochrome c; (2) myoglobin; (3) chymotrypsinogen; (4) ovalbumin; (A) dimeric flavodoxin; and (B) monomeric flavodoxin.

mately 42,000. The SzO,, for Peak B flavoprotein was found to be 2.3. Using both the diffusion coefficient (9.02 x 10-7) and the partial specific volume (0.72) reported for the Shethna flavoprotein (211, the molecu0

FLAVODOXIN

329

lar weight of Peak B flavoprotein was calculated to be 23,000, in good agreement with the Sephadex data (Fig. 2). The molecular weight of the flavoprotein in Peak A is 10-G% less than would be expected for the flavodoxin dimer, possibly because of the use of an incorrect partial specific volume in the calculation. The partial specific volume was assumed to be the same as that reported for the monomeric A. vineZandii flavodoxin (21). Although the molecular weight of the Peak A flavoprotein as determined by sedimentation velocity centrifugation was lower than expected for dimeric flavodoxin, treatment of this high molecular weight flavoprotein (or the apoprotein prepared from Peak A flavoprotein) with 0.2% SDS + 0.2% 2-mercaptoethanol for 2 h at 37” resulted in a single product whose molecular weight (determined by sedimentation velocity centrifugation) was approximately equivalent to that of monomeric flavodoxin. Additional evidence of a monomer-dimer relationship in solutions of flavodoxin was obtained from SDS gel electrophoresis. The high and low molecular weight flavoprotein species (after treatment with SDS and 2-mercaptoethanol) migrated on SDS gels at the same rate (Fig. 3, gels a

b

C

+ FIG. 3. SDS gel electrophoresis of dimeric and monomeric Azotobacter flavodoxin (azotoflavin). Flavoproteins were obtained as Peak A and Peak B from Sephadex column chromatography (see Fig. 2). Gel (a) 25 pg flavoprotein from Peak A (dimeric flavodoxin; gel (b) 25 fig flavoprotein from Peak B (monomeric flavodoxin); gel (cl a mixture of 15 pg each of flavoprotein from Peaks A and B.

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and b). Furthermore, a mixture of these two SDS-treated flavoproteins produced on disc gels a single band (Fig. 3, gel c) whose Rfwas identical to those of gels a and b. Homogeneous solutions of monomeric and dimeric Azotobacter flavodoxin have identical AZT4:A 452absorbance ratios, that is, identical ratios of protein to prosthetic group, suggesting that dimerization is a simple aggregation of two flavoprotein molecules. The reported effect of 2-mercaptoethanol on this flavoprotein (13) suggests that protein aggregations may be the result of an intramolecular disulfide bond between two flavoprotein subunits. To test this idea, a mixture of monomeric and dimeric flavodoxin was chromatographed on Sephadex in the presence and absence of 2-mercaptoethanol (Fig. 4). In the absence of 2-mercaptoethanol, this mixture of monomeric and dimeric flavodoxin separated into two fractions on Sephadex (elution pattern

C. YOCH

shown by closed circles); the two flavoprotein fractions were then recombined and chromatographed again on the same column, which had now been equilibrated with buffer containing 10 mM 2-mercaptoethanol. The elution pattern (open circles) of the protein chromatographed in the presence of 2-mercaptoethanol showed it to be predominantly in the low molecular weight (monomeric) form (Fig. 4). These results are consistent with the interpretation that 2-mercaptoethanol reduced the disulfide group of the dimer back to the original cysteinyl sulfhydryl groups, resulting in the conversion of dimer to monomer. Sodium dithionite was ineffective in converting the dimer to the monomer. Because Azotobacter flavodoxin has but one cysteinyl residue (21), there should be no free sulfhydryl groups in a solution of the dimer. Attempts to show a difference in reactivity of the monomer and dimer to the sulfhydryl binding reagent p-chloromercuribenzoate (PCMB) were unsuccessful as the sulfhydryl group of the monomer was also inaccessable to the mercurial, even in the presence of 4 M guanidine. Effect of Dimerization on Some Oxidation -Reduction Properties of Azotobacter Flavodoxin (Azotofluvin)

60

70 Effluenf

80 fm/l

90

100

FIG. 4. Sephadex G-100 column chromatography of monomeric and dimeric Azotobacter flavodoxin in the presence and absence of 2mercaptoethanol. The column (1.5 x 85 cm) was equilibrated with 20 mM phosphate buffer (pH 7.4), charged with 2.0 ml of a flavoprotein solution containing about equal amounts of monomer and dimer, and eluted with buffer at a flow rate of 1.8 ml/h. After these proteins eluted, the column was equilibrated with about 100 ml of buffer containing 10 mM 2-mercaptoethanol. The monomeric and dimeric proteins were recombined, concentrated to 2.0 ml with a Diaflo ultratiltration cell, put on the column, and eluted with buffer containing 2-mercaptoethanol. Chromatography in presence (0) and absence (0) of S-mercaptoethanol.

If dimerization of Azotobacter flavodoxin is a result of disulfide bonding between the cysteine sulfhydryl group of two molecules, the influence this sulfhydryl group has on the oxidation-reduction properties of the flavoprotein might be examined by comparing these properties of the monomer, which has a free cysteinyl sulfhydryl group, and the dimer, which no longer has a free sulthydryl group. The kinetics of anaerobic dithionite reduction of monomeric and dimeric flavodoxin are shown in Fig. 5. The oxidized monomer and dimer in 25 mM phosphate buffer @H 7.0) were reduced by a sevenfold excess of sodium dithionite first to the semiquinone form of the flavoprotein. (This form has a characteristic absorption band at 580 nm; therefore, an increase in absorbance at 580 nm indicates the formation of the semiquinone.) The rate of reduction of the oxidized monomer to the semiquinone formwas two

DIMERIZATION

OF AZOTOBACZ’ER

331

FLAVODOXIN

While the rate of reduction of monomer and dimer differ significantly, the rate of semiquinone oxidation by oxygen is essentially identical in these two species (Fig. 6), indicating that the cysteinyl sulfhydryl group does not affect the ability of oxygen to remove an electron from the flavin ring.

FIG. 5. Anaerobic reduction ofAzotobacter flavodoxin monomer and dimer by sodium dithionite. The reaction mixtures contained (in 2.0 ml): Flavoprotein, 0.22 pmoles, and phosphate buffer (pH 7.0), 50 pmoles. The reaction mixture in a stoppered Thunberg cuvette was evacuated and refilled with highpurity argon live to six times. A sevenfold excess of sodium dithionite was added to the protein solution in the cuvette anaerobically with a syringe. The cuvettes were opened to air and shaken at the point indicated by the arrow CT).

to four times greater than was the rate of reduction of the dimer. Under these conditions, both monomer and dimer were reduced beyond the semiquinone form until more than half of the flavoprotein was in the fully reduced (hydroquinone) form (indicated by the subsequent decrease in absorbance at 580 nm). Variation in pH between 6.8 and 7.4 had little or no effect on the pattern of flavoprotein reduction. When the cuvettes were opened to air (as indicated by the arrow in Fig. 5), the hydroauinone form of both the monomer and the dimer was rapidly reoxidized to the semiquinone form (as indicated by the rapid increase in absorbance at 580 nm). The fact that the absorbance at 580 nm exceeds that observed during the reduction process indicates that some of the semiquinone was being reduced to the colorless hydroquinone before all of the flavoprotein was in the semiquinone form. The substantial difference between the monomer and dimer in rate of dithionite reduction suggests that the cysteinyl sulfhydryl group may affect the redox properties of Azotobatter flavodoxin.

Effect of Dimerization on Biological Activity A further indication of the effect of dimerization on the flavoprotein is the lack of biological activity of dimeric Azotobacter flavodoxin. Monomeric flavodoxin transfers electrons from illuminated chloroplasts to the nitrogenase of A. vinelandii, whereas the dimer is completely inactive in this reaction (Fig. 7). The previous observation that low concentrations of methyl viologen (1 PM) greatly stimulated this flavodoxin-mediated “chloroplast-nitrogenase” reaction (14) has been confirmed in this study. The addition of 1 PM methyl viologen to the various concentrations of dimer tested resulted in substantial rates of nitrogenase activity (Fig. 7). Controls with methyl viologen alone at this low concentration showed activities of less than 5 nmoles of ethylene formed per minute. The mechanism of this “methyl viologen effect” on flavodoxin-coupled nitrogenase activity has not been investigated. If dimerization is the result of the I

I

,

,

I

I

,

,

1

I

I

06

+

,

2

4

, 6

,

, 8

,

,< , IO

hours

FIG. 6. First-order

rate plot of Atotobacter flavodoxin monomer and dimer semiquinone oxidation by oxygen. Monomer, 0.095 pmoles/ml, and dimer, 0.105 pmoles/ml. Both flavoproteins were in solution in 25 mM phosphate buffer (pH 7.0).

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Azotabacter

flavodoxin

/mgl

FIG. 7. Effectiveness of Azotobacter flavodoxin (axotoflavin) monomer and dimer in coupling reducing power between illuminated chloroplasts and A. uinehzndii nitrogenase. The reaction mixtures all contained (in 1.5 ml) DEAE-cellulose treated A. uinekzndii nitrogenase extract (6.3 mg of protein), chloroplasts (300 pg of chlorophyll), and the following (pmoles): ascorbate, 10; 2,6-dichlorophenol indophenol, 0.05; N-2-hydroxyethylpiperasine-N’-2ethanesulfonic acid (HEPES) buffer (pH 7.4), 50; MgCl,, 5; creatine phosphate, 40; ATP, 4.0; and creatine phosphokinase, 0.005 mg. Light intensity, saturating; gas phase, 73% argon + 27% acetylene; reaction time, 20 min. (A) dimeric flavodoxin; (0) monomeric flavodoxin; (0) dimer + 1 PM methyl viologen; (A) monomer + 1 PM methyl viologen.

participation of cysteinyl residues in disulfide bridges between subunits, this free sulfhydryl group plays an important role in the transfer of electrons between chloroplasm and nitrogenase. DISCUSSION

Evidence has been presented that Azotoback flavodoxin can exist in either a monomeric or a dimeric form. A number of other electron carrier proteins, including cytochromes (ref. 27 and references therein) and ferredoxins (28,291 have previously been reported to exist in polymeric form after storage. This seems to be the first observation of a similar phenomenon associated with a flavoprotein electron carrier. It is difficult to determine the extent to which dimerization of Azotobacter flavodoxin may occur in uiuo. In freshly isolated preparations, less than 5% of the flavo-

C. YOCH

doxin is in the dimeric form; however, it has not been determined if this is a reflection of what occurs in the cell or is simply the amount formed during the isolation procedure. Although dimerization to the extent reported in this study was the result of prolonged storage, one might observe higher concentrations of flavodoxin dimer in preparations made from oxygenshocked cultures or from cells harvested in the stationary growth phase. Dimerization of Azotobacter flavodoxin appears to be the result of an intramolecular disulfide bridge between the cysteinyl sulfhydryl groups of two molecules. The fact that this cysteinyl sulfhydryl group in the monomer was inaccessible to PCMB suggests that it is buried within the molecule. Under oxidative conditions (such as those encountered in storage), this sulfhydry1 group may become uncovered and form disulfide bonds with other molecules, i.e., dimerization. The effect of dimerization on the oxidation-reduction characteristics and biological activity of Azotobacter flavodoxin suggests the involvement of the cysteinyl residue in the electron transport functions of this flavoprotein. In this regard, the loss of biological activity reported by Van Linn and Bothe (9) after treatment of Azotobacter flavodoxin with ferricyanide might be explained by an oxidation of the cysteinyl sulfhydryl group resulting in the biologically inactive dimer. Although the amino acid composition of azotoflavin (Yasunobu and Yoch, unpublished observations) was the same as that of the Shethna flavoprotein (Azotobacter flavodoxin) (211, some of the redox properties of this flavoprotein differ from those of other Azotobacter strains. First and most striking is the observation that the monomer is reduced well beyond the semiquinone form to a mixture of semiquinone and hydroquinone at a neutral pH (Fig. 51, contrasting with the observations of others that Azotobacter flavodoxin is particularly resistant to reduction beyond the semiquinone form (4, 9, 22, 23). Other differences in redox properties between this flavoprotein and those of other Azotobacter strains are: (i) the rate of dithionite-induced semiquinone formation (Fig. 5) is approxi-

DIMERIZATION

OF AZOTOBACTER

mately four to five times faster than that previously reported (4, 22, 23); (ii) the rate of semiquinone oxidation by oxygen (Fig. 6) is about twice that reported by Edmondson and Tollin (23); (iii) the redox potential of the oxidized-semiquinone couple of this flavodoxin is approximately -270 mV (14, 24), whereas Barman and Tollin (25) reported a value of +50 mV for this redox couple; and (iv) Van Linn and Bothe (9) reported that Azotobacter flavodoxin coupled electron flow between illuminated chloroplasts and NADP but similar attempts with the flavodoxin from our strain of Azotobacter were unsuccessful (see ref. 26). Although these differences do not seem to be reflected in the amino acid composition, they may be a reflection of amino acid sequence changes. ACKNOWLEDGMENTS I thank Dr. Dale Edmondson for helpful discussions during the course of this study and Mr. William Ufert for technical assistance. This investigation was aidedin part by NSF Grant BMS71-01204 to D. I. Arnon. Note added in proofi Dr. G. Tollin of the University of Arizona has confirmed the finding reported herein that the flavodoxin from our strain of Azotobutter uinelundii is reduced beyond the semiquinone stage at pH 7, which makes it unlike the flavodoxins from other strains of Azotobucter. He further observed that both the rate of photoreduction and 0, oxidation of this flavodoxin were approximately twice that of the flavodoxin from his A. uinelandii strain 0 (from Wisconsin). Because the flavodoxins from all other strains of A. vinelundii (including strain OP) have been reported to have similar redox characteristics (unlike those of our strain), the bacterium used in this study will be referred to as A. uinelundii strain OP (Berkeley) in future studies. REFERENCES 1. SHETHNA, Y. I., WILSON, P. W., HANSEN, R. E., AND BEINERT, H. (1964) Proc. Nut. Acud. Sci. USA 52, 1263-1271. 2. SHETHNA, Y. I., WILSON, P. W., AND BEINERT, H. (1966) Biochim. Biophys. Actu 113, 223234. 3. BULEN, W. A., LE COMTE, J. R., BURNS, R. C., AND HINKSON, J. (1965) in Iron-Sulfur Proteins: Role in Energy Conversion (San Pietro, A., ed.), pp. 261-274, Antioch Press, Yellow Springs, Ohio. 4. HINKSON, J. W., AND BULEN, W. A. (1967) J. Biol. Chem. 242, 3345-3351.

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5. BENEMANN, J. R., YOCH, D. C., VALENTINE, R. C., AND ARNON, D. I. (1969) Proc. Nut. Acad. Sci. USA 64, 1079-1086. 6. YOCH, D. C., BENEMANN, J. R., VALENTINE, R. C., AND ARNON, D. I. (1969) Proc. Nut. Acud. Sci. USA 64, 1404-1410. 7. YOCH, D. C., AND ARNON, D. I. (1972) J. Biol. Chem. 247, 4514-4520. 8. BENEMANN, J. R., AND VALENTINE, R. C. (1972) Advan. Microbial. Physiol. 8, 59-104. 9. VAN LINN, B., AND BOTHE, H. (1972)Arch. Mikrobiol. 82, 155-172. 10. HARDY, R. W. F., AND BURNS, R. C. (1973) in Iron-Sulfur Proteins (Lovenberg, W., ed.), Vol. 1, pp. 65-110, Academic Press, New York. 11. MAC KNIGHT, M. L., GRAY, W. R., AND TOLLIN, G. (1974) Biochem. Biophys. Res. Commun. 59,630-637. 12. KNIGHT, E., JR., D’EUSTACHIO, A. J., AND HARDY, R. W. F. (1966) Biochim. Biophys. Actu 113, 626-628. 13. HINKSON, J. W. (1968) Biochemistry 7, 26662672. 14. YOCH, D. C. (1972) Biochem. Biophys. Res. Commun. 49, 335-342. 15. SCH~LLHORN, R., AND BURRIS, R. H. (1966) Fed. Proc. 25, 710. 16. DILWORTH, M. J. (1966) Biochim. Biophys. Actu 127, 285-294. 17. HARDY, R. W. F., HOLSTEN, R. D., JACKSON, E. K., AND BURNS, R. C. (1968) PZuntPhysioZ. 43, 1185-1207. 18. ANDREWS, P. (1965) Biochem. J. 96, 595-606. 19. DAVIS, B. J. (1964) Ann. N. Y. Acud. Sci. 121, 404-427. 20. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 244, 4406-4412. 21. EDMONDSON, D. E., AND TOLLIN, G. (1971) Biochemistry 10, 124-132. 22. SHETHNA, Y. I., BEINERT, H., AND HEMMERICH, P., unpublished data cited by Hemmerich, P., Veeger, C., and Wood, H. S. C. (1965)Agnew. Chem. Znt. Ed. 4, 671-689. 23. EDMONDSON, D. E., AND TOLLIN, G. (1971) Biochemistry 10, 133-145. 24. YOCH, D. C. (in press 1975) in Dinitrogen Fixation (Hardy, R. W. F., gen. ed.) J. Wiley. 25. BARMAN, B. G., AND TOLLIN, G. (1972) Biochemistry 11,4755-4759. 26. Yom, D. C. (1974) J. Gen. Microbial. 83, 153164. 27. SINGH, J., AND WASSERMAN, A. R. (1971) J. Biol. Chem. 246,3532-3541. 28. KERESZTES-NAGY, S., AND MARGOLIASH, E. (1966) J. Biol. Chem. 241, 5955-5966. 29. GERSONDE, K., TRITTELVITZ, E., SCHLAAK, H.E., AND STABEL, H.-H. (1971)&r. J. Biochem. 22, 57-65.