VFe Protein Research Articles

Understanding the P-cluster of Vanadium Nitrogenase: an EPR and XAS study of the holo vs. apo forms of the enzyme.

The catalytic moiety of nitrogenases contains two complex metalloclusters: the M-cluster (also called cofactor), where the catalytic reduction of substrates takes place, and the [Fe8S7] P-cluster responsible for electron transfer. Due to discrepancies between crystallography and EPR spectroscopy, the exact structure of the P-cluster in the VFe protein remains a topic of debate. Herein, we use an apo-form of VFe (which retains the P-cluster but lacks the FeVco) to study the VFe P-cluster. SDS-PAGE and NativePAGE showed a heterogeneous composition of the VFe and the apo-VFe samples with presence of α1β2δ2 and α1β2 complexes. The parallel mode EPR measurements of IDS oxidized MoFe, apo-MoFe, and VFe samples reveal a signal at g = 12 associated with the two-electron oxidized state of the P-cluster (P2+) for all three samples, albeit with different intensities. In contrast, no P2+ was observed for IDS oxidized apo-VFe. Additionally, comparisons between apo-MoFe, apo-VFe and the model complex (NBu4)2[Fe4S4(SPh)4] via XAS and EXAFS measurements showed that apo-VFe does not contain a fully formed [Fe8S7] P-cluster, but rather is comprised of fragmented iron-sulfur clusters. Our results point to a possible variation on the structure of the P-cluster in the different forms of the nitrogenase.

Light-driven Transformation of Carbon Monoxide into Hydrocarbons using CdS@ZnS : VFe Protein Biohybrids.

Enzymatic Fisher-Tropsch (FT) process catalyzed by vanadium (V)-nitrogenase can convert carbon monoxide (CO) to longer-chain hydrocarbons (>C2) under ambient conditions, although this process requires high-cost reducing agent(s) and/or the ATP-dependent reductase as electron and energy sources. Using visible light-activated CdS@ZnS (CZS) core-shell quantum dots (QDs) as alternative reducing equivalent for the catalytic component (VFe protein) of V-nitrogenase, we first report a CZS : VFe biohybrid system that enables effective photo-enzymatic C-C coupling reactions, hydrogenating CO into hydrocarbon fuels (up to C4) that can be hardly achieved with conventional inorganic photocatalysts. Surface ligand engineering optimizes molecular and opto-electronic coupling between QDs and the VFe protein, realizing high efficiency (internal quantum yield >56 %), ATP-independent, photon-to-fuel production, achieving an electron turnover number of >900, that is 72 % compared to the natural ATP-coupled transformation of CO into hydrocarbons by V-nitrogenase. The selectivity of products can be controlled by irradiation conditions, with higher photon flux favoring (longer-chain) hydrocarbon generation. The CZS : VFe biohybrids not only can find applications in industrial CO removal for high-value-added chemical production by using the cheap, renewable solar energy, but also will inspire related research interests in understanding the molecular and electronic processes in photo-biocatalytic systems.

Quantum Mechanics/Molecular Mechanics Study of Resting-State Vanadium Nitrogenase: Molecular and Electronic Structure of the Iron-Vanadium Cofactor.

The nitrogenase enzymes are responsible for all biological nitrogen reduction. How this is accomplished at the atomic level, however, has still not been established. The molybdenum-dependent nitrogenase has been extensively studied and is the most active catalyst for dinitrogen reduction of the nitrogenase enzymes. The vanadium-dependent form, on the other hand, displays different reactivity, being capable of CO and CO2 reduction to hydrocarbons. Only recently did a crystal structure of the VFe protein of vanadium nitrogenase become available, paving the way for detailed theoretical studies of the iron–vanadium cofactor (FeVco) within the protein matrix. The crystal structure revealed a bridging 4-atom ligand between two Fe atoms, proposed to be either a CO32– or NO3– ligand. Using a quantum mechanics/molecular mechanics model of the VFe protein, starting from the 1.35 Å crystal structure, we have systematically explored multiple computational models for FeVco, considering either a CO32– or NO3– ligand, three different redox states, and multiple broken-symmetry states. We find that only a [VFe7S8C(CO3)]2– model for FeVco reproduces the crystal structure of FeVco well, as seen in a comparison of the Fe–Fe and V–Fe distances in the computed models. Furthermore, a broken-symmetry solution with Fe2, Fe3, and Fe5 spin-down (BS7-235) is energetically preferred. The electronic structure of the [VFe7S8C(CO3)]2– BS7-235 model is compared to our [MoFe7S9C]− BS7-235 model of FeMoco via localized orbital analysis and is discussed in terms of local oxidation states and different degrees of delocalization. As previously found from Fe X-ray absorption spectroscopy studies, the Fe part of FeVco is reduced compared to FeMoco, and the calculations reveal Fe5 as locally ferrous. This suggests resting-state FeVco to be analogous to an unprotonated E1 state of FeMoco. Furthermore, V–Fe interactions in FeVco are not as strong compared to Mo–Fe interactions in FeMoco. These clear differences in the electronic structures of otherwise similar cofactors suggest an explanation for distinct differences in reactivity.

Nitrogenase Bioelectrochemistry for Synthesis Applications

The fixation of atmospheric dinitrogen to ammonia by industrial technologies (such as the Haber Bosch process) has revolutionized humankind. In contrast to industrial technologies, a single enzyme is known for its ability to reduce or "fix" dinitrogen: nitrogenase. Nitrogenase is a complex oxidoreductase enzymatic system that includes a catalytic protein (where dinitrogen is reduced) and an electron-transferring reductase protein (termed the Fe protein) that delivers the electrons necessary for dinitrogen fixation. The catalytic protein most commonly contains a FeMo cofactor (called the MoFe protein), but it can also contain a VFe or FeFe cofactor. Besides their ability to fix dinitrogen to ammonia, these nitrogenases can also reduce substrates such as carbon dioxide to formate. Interestingly, the VFE nitrogenase can also form carbon-carbon bonds. The vast majority of research surrounding nitrogenase employs the Fe protein to transfer electrons, which is also associated with the rate-limiting step of nitrogenase catalysis and also requires the hydrolysis of adenosine triphosphate. Thus, there is significant interest in artificially transferring electrons to the catalytic nitrogenase proteins. In this Account, we review nitrogenase electrocatalysis whereby electrons are delivered to nitrogenase from electrodes. We first describe the use of an electron mediator (cobaltocene) to transfer electrons from electrodes to the MoFe protein. The reduction of protons to molecular hydrogen was realized, in addition to azide and nitrite reduction to ammonia. Bypassing the rate-limiting step within the Fe protein, we also describe how this approach was used to interrogate the rate-limiting step of the MoFe protein: metal-hydride protonolysis at the FeMo-co. This Account next reviews the use of cobaltocene to mediate electron transfer to the VFe protein, where the reduction of carbon dioxide and the formation of carbon-carbon bonds (yielding the formation of ethene and propene) was realized. This approach also found success in mediating electron transfer to the FeFe catalytic protein, which exhibited improved carbon dioxide reduction in comparison to the MoFe protein. In the final example of mediated electron transfer to the catalytic protein, this Account also reviews recent work where the coupling of infrared spectroscopy with electrochemistry enabled the potential-dependent binding of carbon monoxide to the FeMo-co to be studied. As an alternative to mediated electron transfer, recent work that has sought to transfer electrons to the catalytic proteins in the absence of electron mediators (by direct electron transfer) is also reviewed. This approach has subsequently enabled a thermodynamic landscape to be proposed for the cofactors of the catalytic proteins. Finally, this Account also describes nitrogenase electrocatalysis whereby electrons are first transferred from an electrode to the Fe protein, before being transferred to the MoFe protein alongside the hydrolysis of adenosine triphosphate. In this way, increased quantities of ammonia can be electrocatalytically produced from dinitrogen fixation. We discuss how this has led to the further upgrade of electrocatalytically produced ammonia, in combination with additional enzymes (diaphorase, alanine dehydrogenase, and transaminase), to selective production of chiral amine intermediates for pharmaceuticals. This Account concludes by discussing current and future research challenges in the field of electrocatalytic nitrogen fixation by nitrogenase.

Electroenzymatic C-C Bond Formation from CO2.

Over the past decade, there has been significant research in electrochemical reduction of CO2, but it has been difficult to develop catalysts capable of C-C bond formation. Here, we report bioelectrocatalysis of vanadium nitrogenase from Azotobacter vinelandii, where cobaltocenium derivatives transfer electrons to the catalytic VFe protein, independent of ATP-hydrolysis. In this bioelectrochemical system, CO2 is reduced to ethylene (C2H4) and propene (C3H6), by a single metalloenzyme.

Characterization of an M-Cluster-Substituted Nitrogenase VFe Protein.

ABSTRACTThe Mo- and V-nitrogenases are two homologous members of the nitrogenase family that are distinguished mainly by the presence of different heterometals (Mo or V) at their respective cofactor sites (M- or V-cluster). However, the V-nitrogenase is ~600-fold more active than its Mo counterpart in reducing CO to hydrocarbons at ambient conditions. Here, we expressed an M-cluster-containing, hybrid V-nitrogenase in Azotobacter vinelandii and compared it to its native, V-cluster-containing counterpart in order to assess the impact of protein scaffold and cofactor species on the differential reactivities of Mo- and V-nitrogenases toward CO. Housed in the VFe protein component of V-nitrogenase, the M-cluster displayed electron paramagnetic resonance (EPR) features similar to those of the V-cluster and demonstrated an ~100-fold increase in hydrocarbon formation activity from CO reduction, suggesting a significant impact of protein environment on the overall CO-reducing activity of nitrogenase. On the other hand, the M-cluster was still ~6-fold less active than the V-cluster in the same protein scaffold, and it retained its inability to form detectable amounts of methane from CO reduction, illustrating a fine-tuning effect of the cofactor properties on this nitrogenase-catalyzed reaction. Together, these results provided important insights into the two major determinants for the enzymatic activity of CO reduction while establishing a useful framework for further elucidation of the essential catalytic elements for the CO reactivity of nitrogenase.

QM/MM calculations reveal a bridging hydroxo group in a vanadium nitrogenase crystal structure.

A new 1.2 Å crystal structure of vanadium nitrogenase, isolated under turnover conditions, recently revealed a light atom ligand (OH or NH) replacing the bridging S2B sulfide of the FeV cofactor. QM/MM calculations on the new structure now reveal the light-atom ligand to be a bridging hydroxo group, probably derived from water binding to the cofactor.

Widening the Product Profile of Carbon Dioxide Reduction by Vanadium Nitrogenase.

Two reaction systems based on vanadium nitrogenase were previously shown to reduce CO2 to hydrocarbons: 1) an enzyme-based system that used both components of V nitrogenase for ATP-dependent reduction of CO2 to ≤C2 hydrocarbons; and 2) a cofactor-based system that employed SmI2 to supply electrons to the isolated V cluster for an ATP-independent reduction of CO2 to ≤C3 hydrocarbons. Here, we report ATP-independent reduction of CO2 to hydrocarbons by a reaction system comprising Eu(II) DTPA and the VFe protein of V nitrogenase. Combining features of both enzyme- and cofactor-based systems, this system exhibits improved C-C coupling and a broader product profile of ≤C4 hydrocarbons. The C-C coupling does not employ CO2 -derived CO, and it is significantly enhanced in D2 O. These observations afford initial insights into the characteristics of this unique reaction and provide a potential template for future design of catalysts to recycle the greenhouse gas CO2 into useful products.

Unique features of the nitrogenase VFe protein from Azotobacter vinelandii

Nitrogenase is an essential metalloenzyme that catalyzes the biological conversion of dinitrogen (N(2)) to ammonia (NH(3)). The vanadium (V)-nitrogenase is very similar to the "conventional" molybdenum (Mo)-nitrogenase, yet it holds unique properties of its own that may provide useful insights into the general mechanism of nitrogenase catalysis. So far, characterization of the vanadium iron (VFe) protein of Azotobacter vinelandii V-nitrogenase has been focused on 2 incomplete forms of this protein: alphabeta(2) and alpha(2)beta(2), both of which contain the small delta-subunit in minor amounts. Although these studies provided important information about the V-dependent nitrogenase system, they were hampered by the heterogeneity of the protein samples. Here, we report the isolation and characterization of a homogeneous, His-tagged form of VFe protein from A. vinelandii. This VFe protein has a previously-unsuspected, alpha(2)beta(2)delta(4)-heterooctameric composition. Further, it contains a P-cluster that is electronically and, perhaps, structurally different from the P-cluster of molybdenum iron (MoFe) protein. More importantly, it is catalytically distinct from the MoFe protein, particularly with regard to the mechanism of H(2) evolution. A detailed EPR investigation of the origins and interplays of FeV cofactor- and P-cluster-associated signals is presented herein, which lays the foundation for future kinetic and structural analysis of the VFe protein.

XAFS studies of nitrogenase: the MoFe and VFe proteins and the use of crystallographic coordinates in three-dimensional EXAFS data analysis.

This paper reports a three-dimensional EXAFS refinement of the Mo coordination sphere of the FeMoco cluster of the dithionite-reduced MoFe protein from Klebsiella pneumoniae nitrogenase (Kp1) using the 1.6 A-resolution crystallographic coordinates. At this resolution, the positions of the heavy (Fe and S) atoms of the cluster are well determined and there is excellent agreement between the crystallographic and EXAFS models. However, the lighter homocitrate and histidine ligands are poorly determined in the crystal structure, and it is shown that the application of EXAFS-derived distance restraints during the early stages of crystallographic refinement provides a means of substantially improving (by approximately 0.1 A) the final crystallographic model. The consistency of the EXAFS analysis with the crystallographic information in this case justifies applications of EXAFS to cases where protein crystal structures are absent. Thus, the VFe protein of V-nitrogenase has been shown by EXAFS to possess a V-atom site catalytically similar to the well characterized MoFe-nitrogenases, with V replacing Mo.

Isolation of two forms of the nitrogenase VFe protein from Azotobacter vinelandii.

When Q-Sepharose was used in the purification of the V nitrogenase proteins from Azotobacter vinelandii, an increase in resolution was observed that resulted in a separation of the nitrogenase component 1 protein (Av1') into two forms, labeled Av1'A and Av1'B. Even though both forms possessed the same enzymatic behavior, Av1'A exhibited a lower specific activity and migrated during gel filtration with an apparent lower molecular weight than Av1'B. Furthermore, SDS-polyacrylamide gel electrophoresis showed different relative compositions of the two major subunits of both forms, with Av1'A possessing a trimer (alpha beta 2) pattern compared to the more typical tetramer (alpha 2 beta 2) pattern found for Av1'B. Metal analysis indicated a V-to-Fe ratio of 1:19 for Av1'A and 1:15 (or 2:30) for Av1'B, while acid-labile sulfide analysis showed that Av1'A possessed about half as much sulfide as Av1'B. EPR spectroscopy revealed that both proteins retained the S = 3/2 and S = 1/2 signals observed in earlier isolations, with an additional S = 1/2 signal present in the spectrum of protein A. These results suggest that Av1'A is an incomplete form of the VFe protein, containing only one cofactor and one P cluster with an additional [Fe4-S4]-like cluster. The presence of a V storage protein in A. vinelandii was also investigated. Although no V storage protein was found, two V-binding proteins were observed.

Oxidative titration of the nitrogenase VFe protein from Azotobacter vinelandii: an example of redox-gated electron flow.

The nitrogenase VFe protein of Azotobacter vinelandii (Av1') has been shown to exist in two forms called Av1'A, which has a primary alpha beta 2 trimeric structure, and Av1'B, which has an alpha 2 beta 2 tetrameric structure [Blanchard, C. Z., & Hales, B. J. (1996) Biochemistry 35, 472-478]. Both forms exhibit S = 5/2 EPR signals in the as-isolated state that may be assigned to 1-equiv-oxidized P clusters (P+). These signals are abolished by enzymatic reduction with the component 2 protein (Av2'). Stepwise oxidative titrations of enzymatically reduced Av1'B result in the restoration of the S = 5/2 P+ signals and the concurrent decrease of the S = 3/2 vanadium cofactor signal. Further oxidation results in the appearance of an integer spin signal assigned to the 2-equiv-oxidized P cluster (P2+). Unlike the analogous signal previously observed in Mo nitrogenase component 1 (Av1), which arises from an excited state, the integer spin P2+ signal in Av1'B originates from a ground-state doublet. Similar oxidative titrations of enzymatically reduced Av1'A show redox behavior dramatically different from that of Av1'B, as monitored by EPR spectroscopy. We observed spectral evidence for a redox-induced intramolecular electron transfer between the reduced P cluster and the oxidized FeV cofactor cluster during the titrations.

Mössbauer characterization of the metal clusters in Azotobacter vinelandii nitrogenase VFe protein.

The VFe protein of alternative nitrogenase, isolated from Azotobacter vinelandii, strain LS15 and designated as Av1', has been investigated by Mössbauer spectroscopy. The Mössbauer spectrum of the dithionite-reduced Av1', recorded at 4.2 K with a 60-millitesla magnetic field applied parallel to the gamma-beam, is a superposition of three spectral components: 1) a complex spectrum (the M component) with magnetic hyperfine structures attributed to the paramagnetic FeV cofactor, 2) a component (the P component) consisting of three quadrupole doublets identifiable as the Fe2+, D, and S doublets similar to those observed for the P cluster pairs in MoFe proteins, and 3) a minor (4% of total absorption) quadrupole doublet attributable to adventitiously bound iron. The observed 4.2-K parameters for the Fe2+ (delta EQ = 2.99 mm/s and delta = 0.64 mm/s), D (delta EQ = 0.75 mm/s and delta = 0.63 mm/s), and S (delta EQ = 1.2 mm/s and delta = 0.65 mm/s) iron sites and their temperature dependence are very similar to those observed for the P cluster pairs in the conventional MoFe proteins. Similar to those of the MoFe protein, strong field spectra indicate that these doublets are associated with a diamagnetic system. Their percent absorption intensities (Fe(2+)/D/S = 13.0:32.2:6.8) determined at 4.2 K after the removal of the contributions from the adventitiously bound iron are comparable to those of the P cluster pairs in MoFe proteins. These observations established that Av1' also contains P cluster pairs that are identical, in both composition and quantity, to those of the MoFe proteins; i.e. each molecule contains two P cluster pairs and each pair is formed by two Fe2+, five D, and one S iron sites. Considering that 52% absorption of the P component corresponding to two 8Fe clusters, the remaining 48% absorption determined for the M component is consistent with two 7Fe-containing FeV cofactors/molecule of Av1'. The fact that both P cluster pairs are found in the diamagnetic states implies that the S = 3/2 and S = 1/2 signals detected in earlier EPR measurements are associated with the FeV cofactor and suggests a spin mixture for the FeV cofactor in the dithionite-reduced Av1'.

Hydrazine is a product of dinitrogen reduction by the vanadium-nitrogenase from Azotobacter chroococcum

During the enzymic reduction of N2 to NH3 by Mo-nitrogenase, free hydrazine (N2H4) is not detectable, but an enzyme-bound intermediate can be made to yield N2H4 by quenching the enzyme during turnover [Thorneley, Eady & Lowe (1978) Nature (London) 272, 557-558]. In contrast, we show here that the V-nitrogenase of Azotobacter chroococcum produces a small but significant amount of free N2H4 (up to 0.5% of the electron flux resulting in N2 reduction) as a product of the reduction of N2. The amount of N2H4 formed increased 15-fold on increasing the assay temperature from 20 degrees C to 40 degrees C. Activity cross-reactions between nitrogenase components of Mo- and V-nitrogenases showed that the formation of free N2H4 was associated with the VFe protein. These data provide the first direct evidence for an enzyme intermediate at the four-electron-reduced level during the reduction of N2 by V-nitrogenase.

Vanadium nitrogenase of Azotobacter chroococcum. MgATP-dependent electron transfer within the protein complex.

The kinetics of MgATP-induced electron transfer from the Fe protein (Ac2V) to the VFe protein (AclV) of the vanadium-containing nitrogenase from Azotobacter chroococcum were studied by stopped-flow spectrophotometry at 23 degrees C at pH 7.2. They are very similar to those of the molybdenum nitrogenase of Klebsiella pneumoniae [Thorneley (1975) Biochem. J. 145, 391-396]. Extrapolation of the dependence of kobs. on [MgATP] to infinite MgATP concentration gave k = 46 s-1 for the first-order electron-transfer reaction that occurs with the Ac2V MgATPAclV complex. MgATP binds with an apparent KD = 230 +/- 10 microM and MgADP acts as a competitive inhibitor with Ki = 30 +/- 5 microM. The Fe protein and VFe protein associate with k greater than or equal to 3 x 10(7) M-1.s-1. A comparison of the dependences of kobs. for electron transfer on protein concentrations for the vanadium nitrogenase from A. chroococcum with those for the molybdenum nitrogenase from K. pneumoniae [Lowe & Thorneley (1984) Biochem. J. 224, 895-901] indicates that the proteins of the vanadium nitrogenase system form a weaker electron-transfer complex.

The vanadium nitrogenase of Azotobacter

The properties of the recently characterized vanadium-dependent nitrogenase system of Azotobacter are reviewed and compared with the better characterized Mo-nitrogenases. Vanadium is present in V-nitrogenase in a vanadium- and iron-containing protein (VFe protein), analogous in structure and function to the MoFe protein of Mo-nitrogenase. The VFe protein contains vanadium in a polynuclear cluster in an environment with iron, sulphur and oxygen (or nitrogen or carbon) as nearest neighbour atoms, very similar to the environment of molybdenum in the iron and molybdenum cofactor (FeMoco) centre of the MoFe proteins. A vanadium-containing cluster can be extracted from the VFe protein by treatment with N-methylformamide (NMF). The NMF extract contains vanadium, iron and sulphur in a 1:6:5 ratio and activates the inactive MoFe protein synthesized by some mutant organisms unable to synthesize FeMoco. This hybrid protein has the characteristic substrate reduction pattern of a VFe protein, in forming some C 2 H 6 from C 2 H 2 . These data are consistent with the NMF extract containing an iron and vanadium cofactor FeVaco, analogous to FeMoco. Although the hybrid protein is active in nitrogenase assays with H + or C 2 H 2 as reducible substrates, N 2 is not reduced, suggesting that specific interactions of the cofactor centre with a polypeptide ligand are lacking. Pre-steady-state kinetics of the ATP-dependent electron-transfer from the Fe protein of V-nitrogenase to a VFe protein are very similar to those of Mo-nitrogenase components, consistent with a common mechanism of substrate reduction. However, at low temperatures N 2 is a more effective substrate for V-nitrogenase compared with Mo-nitrogenase.

The vanadium nitrogenase of Azotobacter chroococcum. Purification and properties of the Fe protein.

1. Nitrogenase activity of a strain of Azotobacter chroococcum lacking the structural genes of Monitrogenase (nifHDK) was associated with a V + Fe-containing protein and an Fe-containing protein [Robson, Eady, Richardson, Miller, Hawkins & Postgate (1986) Nature (London) 322, 388-390; Eady, Robson, Richardson, Miller & Hawkins (1987) Biochem. J. 244, 197-207]. 2. The Fe protein was purified to homogeneity by the criterion of Coomassie Blue staining after electrophoresis in 10% or 17% (w/v) polyacrylamide gels in the presence of SDS. One type of subunit, of Mr 32,000 +/- 2000, was found. 3. The native protein had an Mr of 62,500 +/- 2500 and contained approximately 4 Fe atoms and 4 acid-labile sulphide groups per molecule. The amino acid composition was similar to those of other purified Fe proteins, and, characteristically, tryptophan was absent. The specific activities (nmol of protein/min per mg of protein) when assayed under optimum conditions with the VFe protein from this strain were 1211 for H2 evolution under Ar, 337 for NH3 from N2 formation and 349 for C2H2 reduction. Activity of the Fe protein was O2-labile with a t1/2 of 36 s in air. At low temperatures the dithionite-reduced protein exhibited e.p.r. signals consistent with the presence of both S = 1/2 and S = 3/2 spin states. These signals were similar to those given by other nitrogenase Fe proteins, as were the changes in their line shape that occurred in the presence of MgATP or MgADP. The absorbance spectra showed that an increase in absorption occurred in the visible range on reversible oxidation of the dithionite-reduced protein. The oxidized-minus-reduced epsilon 420 was 6000 M-1.cm-1.

The vanadium-iron protein of vanadium nitrogenase from Azotobacter chroococcum contains an iron-vanadium cofactor.

N-Methylformamide extracts of acid-treated precipitated VFe protein of the V-nitrogenase of Azotobacter chroococcum are yellow-brown in colour and contain vanadium, iron and acid-labile sulphur in the approximate proportions 1:6:5. E.p.r. spectra of the extracts exhibit a weak signal with g values near 4.5, 3.6 and 2.0 characteristic of an S = 3/2 metal-containing centre. The N-methylformamide extracts activated the MoFe protein polypeptides from mutants of nitrogen-fixing bacteria unable to synthesize FeMoco, the active centre of Mo-nitrogenase. The active hybrid protein exhibited the characteristic substrate-reducing phenotype associated with the VFe protein except that it could not reduce N2 to NH3. The above data are interpreted as demonstrating the existence of an iron- and vanadium-containing cofactor, FeVaco, within the VFe protein. It is suggested that nitrogen fixation requires specific interactions between FeVaco or FeMoco and their respective polypeptides. The biosynthesis of these cofactors is discussed.

The vanadium nitrogenase of Azotobacter chroococcum. Reduction of acetylene and ethylene to ethane.

1. The vanadium (V-) nitrogenase of Azobacter chroococcum transfers up to 7.4% of the electrons used in acetylene (C2H2) reduction for the formation of ethane (C2H6). The apparent Km for C2H2 (6 kPa) is the same for either ethylene (C2H4) or ethane (C2H6) formation and much higher than the reported Km values for C2H2 reduction to C2H4 by molybdenum (Mo-) nitrogenases. Reduction of C2H2 in 2H2O yields predominantly [cis-2H2]ethylene. 2. The ratio of electron flux yielding C2H6 to that yielding C2H4 (the C2H6/C2H4 ratio) is increased by raising the ratio of Fe protein to VFe protein and by increasing the assay temperature up to at least 40 degrees C. pH values above 7.5 decrease the C2H6/C2H4 ratio. 3. C2H4 and C2H6 formation from C2H2 by V-nitrogenase are not inhibited by H2. CO inhibits both processes much less strongly than it inhibits C2H4 formation from C2H2 with Mo-nitrogenase. 4. Although V-nitrogenase also catalyses the slow CO-sensitive reduction of C2H4 to C2H6, free C2H4 is not an intermediate in C2H6 formation from C2H2. 5. Propyne (CH3C identical to CH) is not reduced by the V-nitrogenase. 6. Some implications of these results for the mechanism of C2H6 formation by the V-nitrogenase are discussed.

The vanadium nitrogenase of Azotobacter chroococcum. Purification and properties of the VFe protein.

1. Nitrogenase activity of a strain of Azotobacter chroococcum lacking the structural genes for conventional nitrogenase (nifHDK) was separated into two components: an Fe-containing protein and a vanadoprotein. 2. The larger protein was purified to homogeneity by the criterion of electrophoresis of 10% (w/v) acrylamide gels in the presence of SDS. Two types of subunit, of Mr 50,000 and 55,000, were present in equal amounts. 3. The protein had an Mr of 210,000 and contained 2 V atoms, 23 Fe atoms and 20 acid-labile sulphide groups per molecule. The Mo content was less than 0.06 g-atom/mol. All the common amino acids were present, with a predominance of acidic residues. Ultracentrifugal analysis gave a maximum sedimentation coefficient of 9.7 S and a symmetrical boundary at 5 mg of protein X ml-1; dissociation occurred at lower concentrations. The specific activities (nmol of product/min per mg of protein), when assayed under optimum conditions with the complementary Fe protein from this strain, were 1348 for H2 evolution, 350 for NH3 formation and 608 for acetylene reduction. Activity was O2-labile, with a t1/2 of 40 s in air. At low temperatures the dithionite-reduced protein showed e.p.r. signals at g = 5.6, 4.35, 3.77 and 1.93, consistent with an S = 3/2 ground state with an additional S = 1/2 centre giving rise to the feature at g = 1.93. The u.v. spectra of dithionite-reduced and thionine-oxidized protein were very similar. Oxidation resulted in a general increase in absorbance in the visible region. The shoulder at 380 nm in the spectrum of reduced protein was replaced with shoulders near 330 nm and 420 nm on oxidation.