Electron Transport to Nitrogenase PURIFICATION AND CHARACTERIZATION OF PYRUVATE:FLAVODOXIN OXIDOREDUCTASE, THE nifJ GENE PRODUCT*

Pyruvate:flavodoxin oxidoreductase, the nifJ gene product of Klebsiella pneumoniae, was purified to ho- mogeneity. Pyruvate:flavodoxin oxidoreductase, flavodoxin, and nitrogenase components I and I1 are the only proteins required for pyruvate-coupled nitrogenase activity. The physiological source of electrons to nitrogenase in K . pneumoniae is pyruvate. Flavodoxin from Azotobacter vinelandii was only one-third as ef- fective as K . pneumoniae flavodoxin in transferring electrons from pyruvate:flavodoxin oxidoreductase to Azotobacter and Klebsiella nitrogenases. Ferredoxins from aerobic, anaerobic and photosynthetic nitrogen- fixing organisms, as well as benzyl viologen and methyl viologen, were ineffective in coupling pyruvate oxidation to nitrogenase activity. One mol each of ace-tyl-coA, Con, and ethylene are formed by pyruvate- supported acetylene reduction. The enzyme contains 8.0 f 0.6 mol of iron and 6.6 f 0.2 mol of acid-labile sulfide per mol of protein (Mr = 240,000). Pyru-vate:flavodoxin oxidoreductase is irreversibly inactivated by air.

transport to nitrogenase was established with purified protein components.

Materials
ATP, creatine phosphate, dithiothreitol, glutathione, thiamin pyrophosphate, coenzyme A, sodium pyruvate, S-acetyl coenzyme A, NAD, L-malate, glycerol, a-ketoglutaric acid, malic dehydrogenase, citrate synthase, Hyamine hydroxide, Tris base, and deoxyribonuclease I were obtained from Sigma. Creatine phosphokinase was obtained from Miles Laboratories, Elkhart, IN. DEAE-cellulose was a Whatman DE52 (microgranular) product. Sephacryl S-200 and Sephadex G-50 were obtained from Pharmacia Fine Chemicals, Piscataway, NJ. Hydroxylapatite used was Bio-Gel HTP from Bio-Rad Laboratories. Sodium [l-'*C]pyruvate and Bray's solution were purchased from New England Nuclear. The solution of radioactive pyruvate was prepared in 0.01 N HC1. All other chemicals and gases were of analytical grade available commercially.

Methods Bacterial Strains and Growth Conditions-K. pneumoniae strain
M5al (UN) is the wild type. Mutant strains UNllO8 (nifJ4428) and UN3409 (nifF.5520) have been described (11). The basal medium, described previously (5), was used as the minimal medium. Cells were grown anaerobically without shaking for 16-18 h at 30 "C in 250 ml of medium containing 28 mM ammonium acetate. Cells were harvested and resuspended in 500 ml of nitrogen-free medium. After incubation for 1.5 h anaerobically, L-serine was added to a final concentration of 0.4 mM. The cells were incubated for an additional 4.5 h and harvested under an argon atmosphere. The cells were washed by suspending them in Nz-sparged 0.1 M Tris-HC1 buffer, pH 7.4, containing 0.86 mM sodium dithionite, followed by centrifugation. The cell paste was stored at -80 "C under an argon atmosphere. For large scale preparations, 18-liter carboys were used and the cells were harvested with a Sharples centrifuge. The cell paste was frozen in liquid nitrogen and stored at -80 "C in a gas-tight container. Preparation of Crude Extract-All buffers used throughout the purification of pyruvakflavodoxin oxidoreductase were sparged with prepurified nitrogen and contained 20% glycerol unless otherwise specified. These buffers were further deoxygenated by repeated evacuation and flushing with argon purified through a heated copper catalyst. These buffers contained 5 mM dithiothreitol, 1 m M glutathione, 1 mM MgClZ. 0.05 mM thiamin pyrophosphate, and 0.86 mM sodium dithionite, added just before use.
The cells were suspended in 0.1 M Tris-HC1 buffer, pH 7.4 (without glycerol), at a concentration of 1 g of cell paste per 2 ml of buffer containing 5-10 pg of deoxyribonuclease I/ml. The cells were broken anaerobically with a French pressure cell at 16,000 psi and centrifuged at 31,000 X g for 40 min at 0-4 "C under an argon atmosphere. The supernatant solution was transferred to an argon-filled bottle with syringe that had been flushed with argon. The crude extract was stored at -20 'C. The pellets of broken cells were pooled and stored at -20 'C for the isolation of flavodoxin.
Pyruuate:Flauodoxin Oxidoreductase-coupled Nitrogenase Assay-The assays were carried out in 9-ml serum vials (1 ml of reaction mixture) containing the following, unless otherwise specified. 25 mM Tris-HC1 (pH 7.4), 2.5 mM ATP, 20 mM creatine phosphate, 5 mM Electron Transport to Nitrogenase MgC12, 0.2 mg of creatine phosphokinase, and 12 p~ K. pneumoniae flavodoxin were placed in vials, sealed with serum stoppers, and repeatedly evacuated and filled with prepurified argon. Two hundred pl of 25 mM Tris-HC1 (pH 7.4) containing 0.86 mM dithionite was added and the vials were incubated for 15 min to ensure removal of the last traces of oxygen. 10 mM sodium pyruvate, 0.05 mM thiamin pyrophosphate, 2 mM dithiothreitol, 0.4 mM coenzyme A, 50 pg of nitrogenase component I, and 25 pg of nitrogenase component I1 were added and the vials were incubated for 5 min to use up trace amounts of dithionite present. The vials were brought to atmospheric pressure by piercing the serum stoppers with a hypodermic needle. One to ten p1 of pyruvate:flavodoxin oxidoreductase and 0.5 ml of acetylene were injected and the vials were incubated at 30 "C for 15 min in a water bath shaker. The reaction was terminated by injecting 0.1 ml of 4 N NaOH and the ethylene formed was measured with a Packard gas chromatograph with a Porapak N column. Specific activities are expressed as nanomoles of ethylene formed per min per mg of protein.
One unit of pyruvate:flavodoxin oxidoreductase is defined as the amount required to produce 1 nmol of ethylene per min under the assay conditions. All reagents were stored in sealed containers under an argon atmosphere. All additions to sealed serum vials were made with microliter syringes flushed with prepurified argon. Because dithionite is used to protect oxygen-labile components, control assays without pyruvate, coenzyme A, or flavodoxin were carried out with each set of assays to ensure that dithionite has not contributed to any observed activity.
Purification Purification of Flauodoxin-Flavodoxin was purified from the pellets of broken cells of wild type K. pneumoniae remaining after removal of the extract. The pellet (100 g) was suspended in 200 ml of 0.2 M potassium phosphate buffer, pH 7.4, without maintaining anaerobic conditions. Triton X-100 was added to a final concentration of 1% and the suspension was stored at -20 "C for overnight. The suspension was thawed, diluted &fold with distilled water, and centrifuged at 13,000 X g for 40 min. The supernatant solution was applied to a DEAE-cellulose column (5 X 25 cm) equilibrated with 0.025 M Tris-HC1 (pH 7.4). The column was washed with one bed volume of 0.025 M Tris-HC1 buffer, pH 7.4, followed by one bed volume of 0.2 M NaCl in the same buffer. The flavodoxin was then eluted with 0.35 M NaCl in 0.025 M Tris-HC1 (pH 7.4). The fractions were assayed for activity by reconstituting electron flow to nitrogen-' The abbreviation used is: SDS, sodium dodecyl sulfate. ase in the extract of a N i p -mutant (UN3409) with pyruvate as an electron source under the conditions described earlier. NifF-mutant extract was the source of pyruvate:flavodoxin oxidoreductase, component I and component I1 in these assays. Active fractions were diluted with an equal volume of 0.025 M Tris-HC1 (pH 7.4) and applied to a DEAE-cellulose column (2.5 X 30 cm) equilibrated with 0.2 M NaCl in 0.025 M Tris-HCl, pH 7.4. The column was washed with one bed volume of 0.2 M NaCl in 0.025 M Tris-HC1 before eluting flavodoxin with 0.35 M NaCl in the same buffer. The fractions containing flavodoxin were pooled, concentrated in an ultrafiltration cell with a PMlO membrane, and applied to a Sephadex G-50 column (2.5 X 55 cm) equilibrated with 0.025 M Tris-HC1, pH 7.4. The column was eluted at a flow rate of 15 ml/h and 2-ml fractions were collected. The most active fractions were pooled, concentrated, and purified to homogeneity by preparative polyacrylamide gel electrophoresis (5, 12). Reduction and Oxidation of Flauodoxin-Flavodoxin absorption spectra were recorded with Beckman model 25 spectrophotometer in 1 ml of reaction mixture. The experimental cuvette contained 25 mM Tris-HC1 (pH 7.4), 2.5 mM ATP, 30 mM creatine phosphate, 5 mM MgC12, 0.2 mg of creatine phosphokinase, 2 mM sodium pyruvate, 0.01 mM thiamin pyrophosphate, 80 p~ coenzyme A, 0.4 mM dithiothreitol, and 30 p~ flavodoxin from K. pneumoniae. The reference cuvette contained all components except flavodoxin. Cuvettes were sealed with rubber stoppers and repeatedly evacuated and filled with prepurified argon. The spectrum of flavodoxin was recorded against the reference. Fifty pg of nitrogenase component I and 5 pg of pyruvate:flavodoxin oxidoreductase were added to both cuvettes and the spectrum was recorded after 10 min. Twenty-five pg of nitrogenase component I1 was added to both of the cuvettes and the spectrum was recorded after 10 min.
Determination of 14C02-14C02 formed from [ l-14C]py~vate was assayed in a 13.5-ml serum vial with a center well (13). The outer well contained 1 ml of reaction mixture for pyruvate-supported nitrogenase activity (mentioned earlier), except that the pyruvate concentration was decreased to 1.8 mM and 0.2 mM [l-'*C]pyruvate was included in the assay. The center well contained 0.2 ml of Hyamine hydroxide solution. Pyruvate-coupled nitrogenase assays were carried out with varying concentrations of pyruvate:flavodoxin oxidoreductase for 15 min at 30 "C in a water bath shaker. The control assay contained all components except pyruvate:flavodoxin oxidoreductase. One-tenth ml of 20% perchloric acid was injected to the outer well to stop the reaction and the vials were incubated in the shaker for 20 min. To assure complete removal of "CO, formed, 0.2 ml of 1 M NaHC03 was injected to the outer well and the shaking was continued for an additional 20 min. Following ethylene estimations, the Hyamine hydroxide solution was counted in 5 ml of Bray's solution (14), using a Packard scintillation counter.
Determination of Acetyl-CoA-Acetyl-coA was determined with malic dehydrogenase and citrate synthase (13). Pyruvate-supported nitrogenase assays were carried out as described earlier. One-tenth ml of 20% perchloric acid was injected in the vials to stop the reaction. Following ethylene estimations, the vials were centrifuged at 600 X g for 10 min. The supernatant solution was transferred to a centrifuge tube, neutralized with 2 N KOH, and centrifuged again. Two-tenths ml of the supernatant solution was used for the determination of acetyl-coA. Under the assay conditions, 10 nmol of acetyl-coA produced 0.049 absorbance change at a wavelength of 340 nm.
K. pneumoniae and Azotobacter uinelandii nitrogenase components I and I1 were purified as previously reported (15,12). A. uinelandii flavodoxin, obtained as a side product during component I1 purification (121, was further purified by the same method used for the purification of K. pneumoniae flavodoxin. Protein concentrations were determined by the method of Lowry et al. (16), using bovine serum albumin as a standard. Iron (17) and acid-labile sulfide (18) contents were determined by published methods. Standard iron solution was obtained from Fisher. Glass-distilled water was used throughout the purification and acid-cleaned glassware were used where necessary. SDS-polyacrylamide gel electrophoresis was performed by slab-gel modification (19) of the Laemmli method (20), using 10% acrylamide.

RESULTS AND DISCUSSION
The results of a representative purification of pyruvate:flavodoxin oxidoreductase are shown in Table I  activity present in crude extract. SDS-polyacrylamide gel electrophoresis of the enzyme showed a single band upon staining with Coomassie blue (Fig. 1). It can be Seen from this figure that the 120,000-dalton band is present in the wild type K. pneumoniae extract, but is missing from an extract of a NifJ-mutant. This polypeptide was previously demonstrated, with the use of mutants, to be coded by the nifJ gene (2, 9, 10). The identification of purified pyruvakflavodoxin oxidoreductase as the nifJ gene product was further confirmed by reconstituting pyruvate-supported nitrogenase activity in the extract of NifJ-mutant (5, 6). Pyruvakflavodoxin oxidoreductase is extremely sensitive to oxygen. A 5-min exposure to air inactivated the enzyme and addition of dithionite did not restore the activity. In crude extracts, pyruvakflavodoxin oxidoreductase is reasonably stable to freezing and thawing, but the purified enzyme is much more susceptible. The pyruvateflavodoxin oxidoreductase contains 8.0 -C 0.6 mol of iron and 6.6 & 0.2 mol of acid-labile sulfide per mol of active enzyme (Mr = 240,000). The only other purified pyruvakflavodoxin oxidoreductase reported (21) was isolated from Escherichia coli. This enzyme was partially purified, but its metal composition was not investigated. Pyruvakferredoxin oxidoreductase from Clostridium midi-urici (13) contains 6 mol of iron and 3 mol of acid-labile sulfide and, that from Halobacterium M b i u m (22), 7.6 mol of iron and 5.2 mol of acid-labile sulfide. Although malate, formate, pyruvate, NADPH-regenerating system, and glucose 6-phosphate have been used as sources of reducing power for K. pneumoniae nitrogenase in crude extracts (4-6), the purified enzyme failed to show any activity with substrates other than pyruvate. a-Ketoglutarate was ineffective in these assays (Table 11). These data support the idea that pyruvate is the only direct physiological substrate of electrons to nitrogenase in K. pneumoniue. E. coli (21) pyruvakflavodoxin oxidoreductase is ineffective with a-ketoglutarate, whereas H. halobium (22) pyruvakferrodoxin oxidoreductase accepts pyruvate, a-ketoglutarate, and a-ketobutyrate as substrates.
The requirements for pyruvakflavodoxin oxidoreductasecoupled nitrogenase activity are presented in Table 11. Omission of any one of the components of the assay system prevented nitrogenase activity. Pyruvate-coupled nitrogenase activity was stimulated 3-to 4-fold by the addition of thiamin pyrophosphate to crude extracts of K. pneumoniae. Thiamin pyrophosphate was added to all the buffers used for purification of pyruvate:flavodoxin oxidoreductase to protect the enzyme (13), hence we could not test its requirement with purified pyruvakflavodoxin oxidoreductase. It is possible that thiamin pyrophosphate is an enzyme-bound cofactor of pyruvate:flavodoxin oxidoreductase, as reported for pyruvakferredoxin oxidoreductases (13.22). vakflavodoxin oxidoreductase was assayed with a variety of low potential electron carriers (Table 111). The pyruvakflavodoxin oxidoreductase is specific to Klebsiellu flavodoxin. At comparable concentrations, A. uinelandii flavodoxin was only one-third as effective as K. pneumoniae flavodoxin in coupling the reducing power of pyruvakflavodoxin oxidoreductase to nitrogenase. Ferredoxins from aerobic, anaerobic, and photosynthetic nitrogen-fixing organisms failed to couple reducing power of pyruvakflavodoxin oxidoreductase to nitrogenase. Nonphysiological low potential electron carriers like benzvl violoeen and methvl violocren also were ineffective. To test its specificity for electron carrier, the pyru-Ferredoxins, be&l viologen; and methyl viologen, reduced enzymatically, have been shown to donate electrons to nitrogenase (23-25). These results indicate that the reducing power of pyruvate:flavodoxin oxidoreductase can be coupled to nitrogenase only by flavodoxins. In contrast, flavodoxin, ferredoxins, benzyl viologen, and methyl viologen have been reported as electron acceptors for pyruvate:flavodoxin oxidoreductase of E. coli (21). Nonspecificity for electron acceptor also was reported for pyruvate:ferredoxin oxidoreductase of C. aczifi-urici (13) and Anabaena cylindrica (26). Because A. vinelandii flavodoxin was only one-third as effective as K. pneumoniae flavodoxin in transferring electrons from pyruvate:flavodoxin oxidoreductase to K. pneumoniae nitrogenase, we examined its effectiveness in coupling electron flow to A. vinelandii nitrogenase (Table IV). It can be seen from these data that A. vinelandii flavodoxin was less effective than K. pneumoniue flavodoxin in coupling the reducing power of pyruvate:flavodoxin oxidoreductase to A. vinelandii and K. pneumoniae nitrogenase. On the other hand, K. pneumoniae flavodoxin was equally effective in transferring electrons to both nitrogenases. From these results, it seems that the interaction of pyruvate:flavodoxin oxidoreductase with flavodoxins rather than the interaction of flavodoxins with nitrogenase, dictates the observed activity. It will be interesting to examine the effectiveness of flavodoxins from other organisms, like E. coli, in this system. Yoch (4) reported that E. coli flavodoxin transferred electrons from illuminated chloroplasts to the nitrogenase of K. pneumoniae much more effectively than to the nitrogenase ofA. vinelandii. E. coli pyruvate:flavodoxin oxidoreductase reduced various low potential electron acceptors, but all except flavodoxin were unable to mediate the activation of pyruvate formate lyase (21).
The change in flavodoxin spectra from the quinone to the hydroquinone brought about by pyruvate:flavodoxin oxidoreductase is illustrated by absorbance changes in Fig. 2. Nitrogenase then can change the hydroquinone to the semiquinone. These spectral changes demonstrate that pyruvate:flavodoxin oxidoreductase reduces flavodoxin to the hydroquinone state, which gets oxidized to semiquinone by transferring electrons to nitrogenase. Oxidation of chemically

Effectiveness of various low potential electron carriers in pyruvute:flauodoxin oxidoreductase-coupled nitrogenase activity
The assays were carried out for 15 min at 30 "C.   (27). The data presented here demonstrate physiological reduction of flavodoxin by pyruvate:flavodoxin oxidoreductase and its oxidation by nitrogenase.
The stoichiometry of the pyruvate:flavodoxin oxidoreductase-coupled nitrogen fixation was deduced by determining all products of the reaction as mentioned under "Methods." The assays were carried out with varying amounts of PYNvate:flavodoxin oxidoreductase. The results of representative experiments (Table V) show that the reaction yields 1 mol each of ethylene, acetyl-coA, and CO,. These results demonstrate that oxidation of 1 mol of pyruvate by pyruvate:flavodoxin oxidoreductase transfers 2 electrons to flavodoxin which in turn transfers electrons to nitrogenase. Similar stoichiometric studies of the pyruvate:ferredoxin oxidoreductase of C. acidi-urici (13) demonstrated transfer of 2 electrons to benzyl viologen or C. acidi-urici ferredoxin. Experiments carried out with pyruvate:flavodoxin oxidoreductase, inactivated by exposure to air, failed to generate any of the reaction products.
Bogusz et al. (7) eluted their putative nifJ gene product from the hydroxylapatite column with 75 mM potassium phosphate buffer. This resulted in 95% loss of activity and 4-fold drop in specific activity. We observed only trace activity (approximately 5% of the total applied to the hydroxylapatite column) in fractions eluted with 75 mM phosphate buffer. An iron-sulfur protein (containing at least 15 iron atoms based on a molecular weight of the NifJ protein), which co-purifies with the nifJ gene product during Sephacryl column chromatography, was eluted with 75 mM phosphate buffer (Fig.  1). Bogusz et al. (7) reported that the dimeric nifJ product contains about 30 mol of iron and 24 mol of labile sulfur. The  Electron Transport to Nitrogenase nifJ gene product purified by us contains 8.0 & 0.6 mol of iron and 6.6 f 0.2 mol of acid-labile sulfide per mol of protein (M, = 240,000). From our elution profile, activity, iron and acidlabile sulfide data, it seems that they (7) purified an ironsulfur protein and misinterpreted it to be the n i J gene product. Our results explain why Bogusz et al. (7) lost most of the enzyme activity in the hydroxylapatite step of purification. The iron-sulfur protein that co-purifies with the pyruvate:flavodoxin oxidoreductase bands in a position close to that of the pyruvate:flavodoxin oxidoreductase on a polyacrylamide gel. Fig. 1 demonstrates that this iron-sulfur protein (with no pyruvate:flavodoxin oxidoreductase activity) is not the protein that is missing in NifJ-mutant extracts; rather, the protein described in this paper does correlate with the nifJ-coded band. Bogusz et al. (7) reported that the purification of the nifJ gene product removed unknown components required for electron flow to the nifJ protein. Our results clearly demonstrate that no products other than pyruvate:flavodoxin oxidoreductase, flavodoxin, and nitrogenase components I and I1 are required for pyruvate-coupled nitrogenase activity. They (7) reported that phosphate and M$+ interfere in the assay and decrease stability of the nifJ gene product. We observed essentially the same activity with 25 mM potassium buffer (pH 7.4) as with 25 mM Tris-HC1 buffer (pH 7.4) in the assays. We observed better than 90% activity of purified pyruvate:flavodoxin oxidoreductase (in 0.2 M potassium phosphate buffer containing 1 mM MgC1,) after 2 days of storage in dry ice. The relatively low yield of our preparations of pyruvate:flavodoxin oxidoreductase (Table I) is not due to inactivation of the enzyme; rather the less active fractions containing the other iron-sulfur protein were discarded after each step of purification.
Extracts of NifJ-, but not of N i f F mutant strains, can be activated (6)(7)(8)28) in vitro by the iron-molybdenum cofactor (FeMo-co). It is possible that the nifJ gene product may be involved, directly or indirectly, in the biosynthesis of the ironmolybdenum cofactor. It is possible that the low potential iron-sulfur center of pyruvate:flavodoxin oxidoreductase may be involved in some reductive step of FeMo-co biosynthesis. Further investigations with NifJ-mutants and the state of FeMo-co in these mutants should answer this question.