Metalloclusters of the nitrogenases

Abstract

Biological nitrogen fixation, the reduction of dinitrogen to ammonia, is catalysed by nitrogenases. These enzymes are found in relatively few groups of bacteria and until recently it appeared that N2 fixation occurred by a single route involving a molybdenum-containing enzyme. However, it is now clear that Mo is not an essential metal for N2 fixation. The soil bacterium Azotobacter chroococcum has an Mo-containing nitrogenase with properties very similar to those found in many other diazotrophic organisms, but it also has a vanadium-containing nitrogenase which functions in the presence of V if Mo is not available. The closely related species A . vinelandii has, in addition to Mo and V nitrogenases, a third nitrogenase which functions only under conditions where Mo and V levels are very low [l].The three nitrogenase systems of A. vinelandii are genetically distinct, being encoded by different structural genes. These genes for the three nitrogenase systems have been cloned and sequenced and have considerable similarity. The nitrogenases have been purified and shown to have similar requirements for activity, viz. a low-potential electron donor, MgATP and the absence of oxygen. They can each be separated into two essential component proteins. All three have a distinct Fe-containing protein (component 2) which, together with either an MoFe protein, a VFe protein, or a homologous protein which contains Fe but only low levels of Mo or V (component l), make up the active nitrogenases.Studies on the structure and function of nitrogenase proteins have benefitted significantly from information provided from studies on the biochemical genetics of nitrogen fixation. The genetics of nitrogen fixation is complex. In Klebsiella pneumoniae, which is the most intensively studied diazotroph and for which there is no evidence for the presence of additional Mo-independent nitrogenases, the 20 nif' genes required solely for nitrogen fixation and its regulation are clustered on a 23-kb region of the chromosome. The entire n[f gene cluster has been sequenced [2]. In addition to the structural genes of nitrogenase (nif'HDK), genes involved in the activation of nitrogenase Fe protein ( n i j w , iron-molybdenum cofactor (FeMoco) biosynthesis (nijii V3ENH) (see below), electron donation ( n i j l f l , several genes of unknown function and the regulatory genes nif'AL are located in this cluster (see Fig. 1 and [3, 41).A . vinelandii and A . chroococcum have a comparable gene cluster, except that they contain additional open reading frames (ORFs) of unknown function and nifABQ map outside the major nif'gene region. The Azotobacter nitrogen fixation genes associated with Mo-independent nitrogenases have also been cloned and sequenced. They include the structural genes of the V nitrogenase (vnf: vanadium nitrogen fixation) and, in A . vinelandii only, the structural genes of the third nitrogenase (an8 alternative nitrogen fixation). In addition to these structural genes, specific regulatory nif'A-like genes and a reiteration of nif'EN-like ...

In Vitro Synthesis of the Iron-Molybdenum Cofactor and Maturation of the nif-encoded Apodinitrogenase

“…The Specificity for Molybdenum of the in Vitro FeMo-co Synthesis System-Cofactor structures of the three nitrogenases are proposed to be essentially similar with vanadium and iron atoms replacing the molybdenum atom in FeV-co and FeFe-co, respectively (2,21,40). The requirement of the nifB and nifV gene products for the biosynthesis of all three cofactors suggests that certain steps in the biosynthesis of FeMo-co are shared in the biosynthetic pathways of all three cofactors.…”

Redox properties and EPR spectroscopy of the P clusters of Azotobacter vinelandii MoFe protein

“…In the relevant biological systems with large D and E-G D zero-field splitting parameters, the 2S+1 levels are split into a singlet (lo>) and S 'non-Kramers doublets' (llz> to IS2>) in zero field. Non-Kramers EPR signals predominantly originate from the transition between the two levels of the 'non-Kramers doublets' and obey the resonance condition of Eqn (1) [50] : (1) in which hv is the microwave quantum (0.3 cm-' in X-band), d is the zero field splitting of the doublet and g = g,,&os a (where a is the angle between the applied magnetic field B and the molecular z axis).…”

The unusual metal clusters of nitrogenase: structural features revealed by x-ray anomalous diffraction studies of the MoFe protein from Clostridium pasteurianum.

“…The known components of FeMoco include 1 Mo, 6-7 Fe, [6][7][8][9] inorganic S atoms, and 1 molecule of homocitrate; two FeMocos bind per tetramer, accounting for the total Mo and half of the Fe content of MoFe protein. In spite of the fact that it has been extracted, purified, and extensively studied by a variety of techniques, the structure of FeMoco is not known and its properties have not been satisfactorily reproduced in model compounds (10,11).…”

“…The Fe atoms not associated with FeMoco also are collected into atypical clusters. In an earlier model for the structure of MoFe protein they were divided into four Fe4S4 clusters, called "P-clusters," which were assigned spectroscopic and redox properties that are both novel and complex (5)(6)(7). Thus it is likely that all of the metal atoms bound by MoFe protein are associated with metalsulfur clusters of unusual structure and/or environment.…”