In Azotobucter vinelandii MoFe protein the oxidation of the P clusters to the S = 7/2 state is associated with a redox reaction with Em,7 = +90 2 10 mV (vs the normal hydrogen electrode), n = 1. A concomitant redox process is observed for a rhombic S = 1/2 EPR signal with g = 1.97, 1.88 and 1.68. This indicates that both S = 1/2 and S = 7/2 signals are associated with oxidized P clusters occurring as a physical mixture of spin states. The maximal intensity of the S = 1/2 and S = 7/2 signals in the mediated equilibrium redox titration is similar if not identical to that of solidthionine-treated samples. Summation of the spin concentration of the S = 1/2 spin state (0.25 2 0.03 spin/ad2) and the S = 7/2 spin state (1.3 * 0.2 spida2p2) confirms that the MoFe protein has absolutely no more than two P clusters. In spectra of enzyme fixed at potentials around -100 mV a very low-intensity g = 12 EPR signal was discovered. In parallel-mode EPR the signal sharpened and increased >lO-fold in intensity which allowed us to assign the g = 12 signal to a non-Kramers system (presumably S = 3). In contrast with the non-Kramers EPR signals of various metalloproteins and inorganic compounds, the sharp absorption-shaped g = 12 signal is not significantly broadened into zero field, implying that the zero field splitting of the non-Kramers doublet is smaller than the X-band microwave quantum. The temperature dependence of this g = 12 EPR signal indicates that it is from an excited state within the integer spin multiplet. A bell-shaped titration curve with Em,7 = -307 * 30 mV and + 81 2 30 mV midpoint potentials is found for the g = 12 EPR signal. We propose that this signal represents an intermediate redox state of the P clusters between the diamagnetic, dithionite-reduced and the fully oxidized S = 7/2 and S = 1/2 state. Redox transitions of two electrons (-307?30mV) and one electron (+90?10mV) link the sequence S = O*S = 3+(S = 7/2 and S = U2). We propose to name the latter paramagnetic oxidation states of the P clusters in nitrogenase POx1 and POx2, and to retain PN for the diamagnetic native redox state. The magnetic circular dichroism and Mossbauer data on thionine-oxidized MoFe protein have to be re-evaluated bearing in mind that the oxidized P clusters can exist in two redox-states. Finally, an account is given of the EPR spectroscopic properties of S = 9/2 and other systems obtained upon superoxidation of the MoFe protein.Nitrogenase is the biological catalyst for the activation of the dinitrogen molecule in aqueous solution. The enzyme complex consists of two dissociable metalloproteins, the =230-kDa ad2 tetrameric MoFe protein and the homodimeric = 62-kDa Fe protein. Substrate binding, activation and reduction takes place on the MoFe protein, presumably

The genome of Pyrococcus furiosus contains the putative mbhABCDEFGHIJKLMN operon for a 14-subunit transmembrane complex associated with a Ni±Fe hydrogenase. Ten ORFs (mbhA±I and mbhM) encode hydrophobic, membrane-spanning subunits. Four ORFs (mbhJKL and mbhN) encode putative soluble proteins. Two of these correspond to the canonical small and large subunit of Ni±Fe hydrogenase, however, the small subunit can coordinate only a single iron-sulfur cluster, corresponding to the proximal [4Fe±4S] cubane. The structural genes for the small and the large subunits, mbhJ and mbhL, are separated in the genome by a third ORF, mbhK, encoding a protein of unknown function without Fe/S binding. The fourth ORF, mbhN, encodes a 2[4Fe±4S] protein. With P. furiosus soluble [4Fe±4S] ferredoxin as the electron donor the membranes produce H 2 , and this activity is retained in an extracted core complex of the mbh operon when solubilized and partially purified under mild conditions. The properties of this membrane-bound hydrogenase are unique. It is rather resistant to inhibition by carbon monoxide. It also exhibits an extremely high ratio of H 2 evolution to H 2 uptake activity compared with other hydrogenases. The activity is sensitive to inhibition by dicyclohexylcarbodiimide, an inhibitor of NADH dehydrogenase (complex I). EPR of the reduced core complex is characteristic for interacting iron-sulfur clusters with E m < 20.33 V. The genome contains a second putative operon, mbxABCDFGHH'MJKLN, for a multisubunit transmembrane complex with strong homology to the mbh operon, however, with a highly unusual putative binding motif for the Ni±Fe-cluster in the large hydrogenase subunit. Kinetic studies of membrane-bound hydrogenase, soluble hydrogenase and sulfide dehydrogenase activities allow the formulation of a comprehensive working hypothesis of H 2 metabolism in P. furiosus in terms of three pools of reducing equivalents (ferredoxin, NADPH, H 2 ) connected by devices for transduction, transfer, recovery and safety-valving of energy.

The flavodoxins from Azotobacter vinelandii cells grown N2-fixing and from cells grown on NH40Ac have been purified and characterized. The purified flavodoxins from these cells are a mixture of three different flavodoxins (Fld I,11, 111) with different primary structures. The three proteins were separated by fast protein liquid chromatography; Fld I eluted at 0.38 M KC1, Fld I1 at 0.43 M KC1 and Fld I11 at 0.45 M KC1. The most striking difference between the three flavodoxins was the midpoint potential (pH 7.0, 25 "C) of the semiquinone/ hydroquinone couple, which was -320 mV for Fld I and -500 mV for the other two flavodoxins (Fld 11 and Fld 111).All three flavodoxins were present in cells grown on NH40Ac. In cells grown on N2 as N source only Fld 1. and Fld I1 were found. The concentration of Fld I1 was 10-fold higher in N2-fixing cells than in cells grown on NH40Ac. Evidence has been obtained that Fld I1 is involved in electron transport to nitrogenase.As will be discussed, our observation that preparations of Azotobacter flavodoxin are heterogeneous, has consequences for the published data.Flavodoxin from Azotobacter vinelandii was first isolated by Shethna et al. in 1964 [l, The protein consists of a single polypeptide chain with 179 amino acid residues, it contains one FMN and has a relative molecular mass of 19990 [S]. There is one single cysteine residue present which can cause dimerization of two flavodoxin molecules, a process which results in the loss of biological activity [9]. In addition to the 5'-phosphate ester on the FMN, flavodoxin contains 2 mol tightly bound phosphate/mol [lo]. One phosphate group is covalently bound to the protein in a phosphodiester linkage between serine and threonine residues. It has been suggested that the other is an acid-labile phosphate in an acyl phosphate linkage with a protein COOH group [ll]. At pH 8.0 and 25°C the redox potential of the quinone/semiquinone couple (E2) of flavodoxin is -250 mV [12-141. However an anomalous value of + 50 mV was also reported for E2 [15]. The redox potential of the semiquinone/hydroquinone couple ( E l ) is -500 mV The primary function of the flavodoxin in Azotobacter species was suggested to be electron transport to nitrogenase. [12-161.In 1969, Benemann et al. [4, 171 showed that flavodoxin was one of the four factors native to A. vinelandii cells needed for electron transport from NADPH to nitrogenase; however, the reported rate was just a fraction of the activity obtained with dithionite as electron donor. It appeared that the endogenous enzyme system was not capable of reducing flavodoxin effectively beyond the semiquinone state, whereas the hydroquinone form is necessary for nitrogenase activity [18, 191. In fact, completely reduced flavodoxin was found to be a good electron donor for nitrogenase, activities being 50% higher than with dithionite [I9 -211. Furthermore flavodoxin from Azotobacter chroococcurn was shown to be nif specific [=I.What argues against flavodoxin being the unique physiological electron donor for ni...

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1. Evidence is presented that the direct depressing effect of ammonium chloride on nitrogen fixation by Azotobacter vinelandii is due to inhibition of the electron transport system to nitrogenase. Furthermore, we were able to confirm the observation [Houwaard, F. (1979) Appl. Environ. Microbiol. in the press] that ammonium chloride has no short-term effect on nitrogen fixation by isolated bacteroids of Rhizobium leguminosarum.2. By means of the flow dialysis technique it could be demonstrated that in A . vinelandii ammonium is taken up as a cation in response to the A $ and that uptake of ammonium specifically inhibits the flow of reducing equivalents to nitrogenase by lowering the A $ across the cytoplasmic membrane. In A . vinelandii, like in bacteroids, the generation of reducing equivalents at a potential low enough to reduce nitrogenase was found to be extremely sensitive towards changes in A$. At A$ values less than 80 mV, interior negative, no such reducing equivalents are generated, while at a d $ value of 110 mV nitrogenase is supplied optimally with reducing equivalents. The nature of the ammonium transport system in A. vinelandii and its significance as a regulator for the rapid 'switch off/switch on' of nitrogenase activity is discussed.3. Bacteroids of R. leguminosarum did not accumulate ammonium and no effect of ammonium on A $ was observed. On the contrary, it could be demonstrated that bacteroids excrete ammonium in response to the A pH.It has been firmly established that the process of nitrogen fixation by free-living bacteria [l-41 as well as by symbiotic associations [5,6] is inhibited in the presence of fixed nitrogen, especially ammonium. In the free-living nitrogen-fixer Azotobacter two effects of ammonium can be distinguished. Firstly, the energetically expensive nitrogenase reaction is rapidly switched off (short-term effect) [l]. Secondly, the synthesis of nitrogenase is repressed, resulting in a decline of total activity (long-term effect) [2,7]. On the other hand, fixed nitrogen does not affect the nitrogenase activity of isolated bacteroids of Rhizobium leguminosarum [8]. However, when these bacteroids are in symbiosis with the plant host cell, fixed nitrogen affects the nitrogenase activity [5,6].Little is known about the short-term effect of am- enough energy in the form of ATP and reducing equivalents. Since cell-free nitrogenases are insensitive to ammonium concentrations that completely inhibit the nitrogenase activity in whole cells [9,10], it has been suggested that ammonium in Azotobacter depresses the flow of reducing equivalents and/or the supply of ATP to nitrogenase [l, 1 I].In the present paper we investigated the shortterm effect of ammonium chloride on nitrogen fixation by the free-living organism Azotobacter vinelandii and the symbiotic nitrogen-fixer R. leguminosarum. Evidence is presented that electron transfer to nitrogenase in A. vinelandii, like in R. leguminosarum bacteroids [12], is regulated exclusively by the electrical component (A$) of the protonmotive for...

In addition to their g = 1.94 EPR signal, nitrogenase Fe-proteins from Azotobacter vinelandii, Azotobacter chroococcum and Klebsiella pneumoniae exhibit a weak EPR signal with g N 5. Temperature dependence of the signal was consistent with an S= 312 system with negative zero-field splitting, D =-5 f 0.7 cm-l. The m, = f 312 ground state doublet gives rise to a transition with fl= 5.90 and the transition within the excited m,= + l/2 doublet has a split g,"=4.8, 3.4. Quantitation gave 0.6 to 0.8 spin.mol-' which summed with -the spin intensity of the S= l/2 g= 1.94 line to roughly 1 spin/mol. MgATP and MgADP decreased the intensity of the S= 312 signal with no concomitant changes in intensity of the S= l/2 signal.

Thionine-oxidized nitrogenase MoFe proteins from Azotohucter vinelundii. Azotobacter cliroococcurn and Klehsiella pneurnoniae exhibit excited-state EPR signals with g = 10.4, 5.8 and 5.5 with a maximal amplitude in the temperature range of 20-50 K. The magnitude of these effective g values, combined with the temperature dependence of the peak area at g = 10.4 from 12 K to 86 K, are consistent with an S = 7/2 system with spin01 cm-' and g = 2.00. This interpretation predicts nine additional effective g values some of which have been detected as broad features of low intensity at g z 10, z 2.5 and z 1.8. The S = 7/2 EPR is ascribed to the multi-iron exchange-coupled entities known as the P clusters.Quantification relative to the S = 312 EPR signal from dithionite-reduced MoFe protein indicates a stoichiometry of one P cluster per FeMo cofactor. Two possible interpretations for these observations, together with data from the literature, are proposed. In the first model there are two P clusters per tetrameric MoFe protein. Each P cluster encompasses approximately 8Fe ions and releases a total of three electrons on oxidation with excess thionine. In the second model the conventional view of four P clusters, each containing approximately 4Fe, is retained. This alternative requires that following one-electron oxidation, the P clusters factorize into two populations, P, and Pb, only one of which is further oxidized with thionine resulting in the S = 7 / 2 system. Both models require eight-electron oxidation of tetrameric MoFe protein to reach the S = 7/2 state.

1. Several low-potential electron carriers from different sources can be photoreduced by the system 3,l O-dimethyl-5-deazaisoalloxazine/N-tris(hydroxymethyl)methylglycine. The carriers studied were flavodoxin, ferredoxin 1 and iron-sulfur protein I1 from Azotohacter vinelundii and the flavodoxins from Desulfovibrio vulgaris and Peptostreptococcus elsdenii.2. Electron transport to A . vinelandii nitrogenase was studied, employing different preparations of the enzyme: a crude complex; a complex reconstituted from the 0.27 M and 0.38 M NaCl fractions after DEAE-cellulose chromatography; a complex reconstituted from the 0.27 M and 0.38 M NaCl fraction plus iron-sulfur protein I1 purified from the 0.15 M NaCl fraction. Of all photoreduced carriers tested, only flavodoxin hydroquinone from A . vinelundii catalyzes significant electron transport to these complexes.3. The time course of oxidation of substrate-amounts of A . vinelandii flavodoxin hydroquinone by catalytic amounts of crude nitrogenase complex shows three characteristic phases : an initial lag phase (l), a phase with constant rate over a range of redox potentials (2) and a final phase with rapidly declining rate (3). It was shown that the Fe-S protein I1 is responsible for the lag phase; the potential where phase 2 changes into phase 3 is at a higher value in the presence of Fe-S protein 11. Pre-reduction of the enzyme photochemically abolishes phase 1 and causes phase 2 to proceed at a higher rate. The rate in phase 2 can be enhanced also by lowering the 'starting potential' of the flavodoxin hydroquinone/semiquinone couple. 4. A . vinelandii flavodoxin shuttles between its hydroquinone and semiquinone forms during steady-state electron transfer. Over 90 % of the reducing equivalents is recovered in ethylene formed from acetylene. The concentrations of flavodoxin hydroquinone to give half-maximum rate in the acetylene reduction assay is 3 -6 pM. A scheme is proposed for the regulation of electron donation to nitrogenase.It is well established that pyruvate is a main source of reducing equivalents for nitrogenase in many aerobic nitrogen-fixing bacteria, and that electrons are usually transferred from pyruvate dehydrogenase via ferredoxin [l]. Benemann et al. [2] as well as Yoch and Arnon [3] considered ferredoxin a possible candidate as electron donor for nitrogen fixation in Azotohacter, but attempts to demonstrate pyruvate-ferredoxin oxidoreductase activity in extracts have given equivocal results [4].The role of flavodoxin in aerobic nitrogen-fixing organisms is also not certain. As in aerobic organisms it might substitute for ferredoxin under conditions of iron-deficiency. Benemann et al. [2,5] were the first to postulate a role of flavodoxin in nitrogen fixation of Azotobacter. They showed that flavodoxin could couple the reducing power of chloroplast photosystem I to nitrogenase in a cell-free extract. However, the activity obtained amounted to only a fraction of the activity with dithionite as electron donor. In addition, the specificity of the re...