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Nitrogenase is an essential metalloenzyme that catalyzes the biological conversion of dinitrogen ([N.sub.2]) to ammonia (N[H.sub.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: [alpha][[beta].sub.2] and [[alpha].sub.2][[beta].sub.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].sub.2][[beta].sub.2][[delta].sub.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 H2 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.

Biological nitrogen fixation, the conversion of atmospheric [N.sub.2] to N[H.sub.3], is an essential process in the global biogeochemical cycle of nitrogen that supports life on Earth. Most of the biological nitrogen fixation is catalyzed by the molybdenum nitrogenase, which contains at its active site one of the most complex metal cofactors known to date, the iron-molybdenum cofactor (FeMo-co). FeMo-co is composed of 7Fe, 95, Mo, R-homocitrate, and one unidentified light atom. Here we demonstrate the complete in vitro synthesis of FeMo-co from [Fe.sup.2+], [S.sup.2-], Mo[O.sub.4.sup.2-], and R-homocitrate using only purified Nif proteins. This synthesis provides direct biochemical support to the current model of FeMo-co biosynthesis. A minimal in vitro system, containing NifB, NifEN, and NifH proteins, together with [Fe.sup.2+], [S.sup.2-], Mo[O.sub.4.sup.2-], R-homocitrate, S-adenosyl methionine, and Mg-ATP, is sufficient for the synthesis of FeMo-co and the activation of apo-dinitrogenase under anaerobic-reducing conditions. This in vitro system also provides a biochemical approach to further study the function of accessory proteins involved in nitrogenase maturation (as shown here for NifX and NafY). The significance of these findings in the understanding of the complete FeMo-co biosynthetic pathway and in the study of other complex Fe-S cluster biosyntheses is discussed. FeMo-co | nitrogen fixation | nif | Fe-S proteins

A study is conducted to demonstrate that wild-type nitrogenase reduces diazene to ammonia. The findings suggest that the reduction of [N.sub.2] and diazene is inhibited by [H.sub.2].

Synthesis, spectroscopic studies and structures of molybdenum(VI) complexes with homocitrate complexes with homocitrate are reported. Results show that the homocitrate ligand trapped by tetranuclear molybdate coordinates to the molybdenum(VI) atom through alpha-alkoxy and alpha-, beta-carboxy groups.

The migration of H atoms over S and Fe atoms in the reaction domain of FeMo-co, the active site of nitrogenase is described and used to explain mechanistic data on the catalyzed reductions of [N.sub.2] and [C.sub.2][H.sub.2]. The positions of H atoms influence the binding of substrates [N.sub.2] and [C.sub.2][H.sub.2] and the bound substrate blocks further migration of H atoms past the binding site.

The Mo-nitrogenase-catalyzed reduction of both cyanide and azide results in the production of excess NH(sub 3), which is an amount of NH(sub 3) formed from the reactions. Unlike Mo-nitrogenase, CO could not completely eliminate either cyanide or azide reduction by V-nitrogenase, but eliminate the inhibition of both electron flux and MgATP hydrolysis by CN(super -).

The existence of a three-member electron-transfer series of single cubanes [(Tp)MoFe3S4](super z) (z = 3-, 2-, 1-) and a four-member series of double cubanes [(Tp)(sub 2)Mo2Fe6S8L4](super z) (z = 4-, 3-, 2-, 1-) with L = F(super -), Cl(super -), N3, PhS(super -) is showed by electrochemical methods, cluster synthesis and X-ray structure determinations. The synthesis and structure of [(Tp)(sub 2)Mo2Fe6S8F2.H2O](super 3-), a new topological analogue of the P(super N) cluster of nitrogenase, is presented (Tp = hydrotris(pyrazolyl)borate(1-)).

The expression of nitrogenase genes of Herbaspirillum sp. B501 associated in shoot of wild rice, Oryza officinalis, is explored by means of reverse transcription-PCR (RT-PCR) targeted at the nifH gene. The results indicate that in planta nitrogen fixation by the endophyte shows a daily rhythm determined by the plant's light environment.

The molecular diversity of the nifH gene is characterized by nested PCR amplification using degenerate primers, followed by cloning and sequencing, in order to understand the structure of marine diazotrophic communities in the tropical and subtropical Atlantic Ocean. The results have shown that some diazotrophic phylotypes, specifically unicellular group A, might be common between the Pacific and Atlantic Oceans.

H ENDOR data from the S(sub EPR1) intermediate formed during turnover of the nitrogenase alpha-195 (super GLN) MoFe protein with C2H2/C2D2 in H2O/D2O buffers, reexamined in context with the study of the intermediate formed from propargyl alcohol, indicate that S(EPR1) is a product complex, likely with C2H4 bound as a ferracycle to a single Fe of the FeMo-cofactor active site. It exhibits the same valencies as those of the CO-bound cofactor of the lo-CO intermediate formed during turnover.

The issue of the existence of a family of molecular iron-nitride clusters of higher nuclearity is investigated. The reason for the investigation is to ascertain whether their features would be advantageous in the interpretation of cofactor structural and electronic properties.

Stopped-flow FTIR spectroscopy is used to monitor continuously the pre-steady- and steady-state phases of azide reduction by nitrogenase and the accompanying hydrolysis of ATP. Exploration of the steady-state time course for azide reduction to zero time showed that one azide binds within 200 ms to each FeMo cofactor.

A research conducted explains the synthesis of the clusters of nitrogenase and a new route is devised to the P cluster structure in molecules. The synthesis and properties of the molybdenum cluster are further discussed.

The synthesis of Mo/Fe/S clusters with the MoFe3S3 core in attempts to model the FeMo-cofactor in nitrogenase is detailed. The [MoFeS4](super 4) cuboidal subunits are reduced by one electron relative to the known [MoFe3S4](super 3) clusters and show shorter Fe-Fe and Fe-Mo distances, and the storage of reducing equivalents into M-M bonds and their use in the reduction of substrates may account for the presence of apparently extensive M-M bonding in the nitrogenase cofactor.

Nitrogenase function in terms of crystal structures of both Fe protein and MoFe protein is described. The isolated FeMoco cluster has low DFT energy and lowest energy when interaction with the protein and solvent environment is included.

Hydrogen production by Anabaena sp. strain 7120 is increased by mutating six nitrogenase amino acid residues predicted to reside within the iron-molybdenum cofactor (FeMo-co) in order to direct electron flow selectively toward proton reduction in the presence of [N.sub.2]. The results have shown the feasibility of engineering cyanobacterial strain for enhanced photobiological production of [H.sub.2] in an aerobic, nitrogen-containing environment.

The isolation and characterization of a fully active species of FeVco from Azotobacter vinelandii which are homologous metal centers with distinct catalytic properties are reported. The study results provide the first EXAFS-based structural model of the isolated FeVco and lay a foundation for future catalytic and structural studies of this unique metallocluster.

Crystal structures with the nitrogenase complex in different nucleotide-bound states have shown major conformational changes that have provided a structural underpinning to suggestions that intercomponent electron transfer (ET) is gated by conformational changes of the complex and of its component protein. The gating hypothesis is tested with studies of the intracomplex oxidation of [Fe.sup.red] by MoFe protein in the presence of a variety of solutes.

The intermediate generated at a high concentration during reduction of the natural nitrogenase substrate ([N.sub.2]) by wild-type MoFe protein has shown that it contains [N.sub.2] bound to the active-site FeMo cofactor. The single type of [super 15]N-coupled nucleus from the field dependence, along with the absence of an associated exchangeable [super 1]H ENDOR signal, is shown to be consistent with the [N.sub.2] molecule bound end-on to the FeMo cofactor.

The carbene cluster [[Fe.sub.4][S.sub.4][([Pr.sup.i.sub.2]NHC[Me.sub.2]).sub.4]] as an accurate structural and electronic analogue of the fully reduced cluster of the iron protein of Azotobacter vinelandii nitrogenase, including a common S = 4 ground state and the issues related to the existence and magnetism of other [[[M.sub.4][S.sub.4][L.sub.4]].sup.2] clusters are demonstrated. The octanuclear clusters which exhibited rhomb-bridged noncubane (RBNC) structure and the clusters with the [Fe.sub.8][S.sub.8] core that possessed edge-bridged double-cubane (EBDC) stereochemistry are two structural solutions for the [M.sub.8][S.sub.8] core in the form of topological isomers whose stability might depend on valence electron count.

The roles of the Fe protein and 16 magnesium ATP (MgATP) hydrolysis and insights gained from success in trapping substrates and inhibitors at the active-site metal cluster FeMo cofactor are analyzed. Nitrogenase is the molybdenum (Mo)-dependent enzyme and the reduction of [N.sub.2] by this enzyme has involved the transient interaction of two component proteins.

The synthesis and characterization of a class of new nitrogenases model complexes [Cp*Fe[([mu]-S[R.sup.1]).sub.2]([mu]-[R.sup.2]N[double bond]NH)FeCp*] ([R.sup.1] = Me, Et; [R.sup.2] = Me, Ph; Cp* = [[eta].sup.5]-[C.sub.5][Me.sub.5]) are described along with their excellent catalytic N-N bond cleavage of hydrazines on diiron centers under ambient conditions. The results have shown that some steps of the biological [N.sub.2] reduction have taken place at diiron sites.

The studies about the molybdenum-independent nitrogenases diazotrophs isolated from wood chip mulch, soil, 'paraffin dirt', and sediments from mangrove swamps are described. Most of the isolates are found to be members of gamma subdivision of the class Proteobacteria and appear to be specifically related to fluorescent pseudomonads and azotobacteria.

The structure of NifB-co, an Fe-S cluster produced by the enzyme NifB, bound to NifX protein from Azotobacter vinelandii is studied. The results revealed that the interstitial light atom is inserted at the NifB-co stage in iron molybdenum cofactor (FeMo-co) biosynthesis.

A useful strategy to improve sustained photobiological hydrogen production in cyanobacteria is demonstrated. The process involves modification of two homocitrate synthase genes, nifV1 and nifV2, from the cyanobacterium Nostoc sp. strain PCC 7120.

An argument that the first introduction of h is via a water chain terminating at water 679 to one of the mu3-S atoms is presented. Using validated density functional calculations of a full chemical repre4sentation of FeMo-co and its connected residues, more than 80 possibilities for the coordination of up to three H atoms, and H2, and H+H2 on the S2A, Fe2, S2B, Fe6, S3B domain of FeMo-co, which is favored by targeted mutagenesis results is characterized.

The mechanistic chemistry of Fe-S-based clusters is discussed. Emphasis is laid on the reactions that are of most relevance to the role of Fe-S clusters as substrate binding and transformation sites such as substitution, protonation, binding of small molecules and ions, and transformation by coupled electron- and proton-transfer reactions.

Density functional theory (DFT) studies of the nitrogenase, to explore the origin and determine the nature of the central light atom and its importance as a structure-stabilizing feature are presented. The results of these studies and a possible mechanism of nitrogenase function are examined.

The synthetic methods, structures, and selected properties of clusters are highlighted. However, the synthetic challenge posed by the nitrogenase metaloclusters with their intrinsic property remains formidable.

The free-energy transformations that occur in nitrogenase based upon theoretical analysis of structural and electrochemical data is described using electrostatic and thermodynamic analysis. It is observed that electrostatic interactions play an important role in the substitution of MgADP by MgATP upon reduction of the [Fe4Se4]S4(super cys) cluster in the Fe protein.

The possibility of an exchanging N by using electron-nuclear double resonance (ENDOR) and electron spin-echo envelope modulation (ESEEM) spectroscopies is tested to examine the FeMo-co in wild-type. Nitrogenase that consists of the electron-transfer Fe protein and active-site-containing MoFe protein reduces N2 to two NH3 under atmospheric pressure and temperature in a reaction coupled to the hydrolysis of ATP.

The structural and energetic consequences of a central Logan in the femora are investigated by means of density functional calculations. It is shown that nitrogen is the only central ligand, which leads both to an energetically stable structure and to a geometry resembling the one found experimentally.