FeMoco substrate binding site

Reactions of isolated FeMoco have also been used in attempts to identify substrate binding sites. Interpretation of these studies is complicated by the existence of vacant protein ligand binding sites on FeMoco, i.e., the sites where cysteine and histidine bind FeMoco to the protein. It is assumed that in extracted cofactor these sites are occupied by the solvent iV-methylformamide. 19F NMR and x-ray absorption experiments [82] and EPR data [83] have demonstrated that CN and methylisocyanide bind to isolated FeMoco. The EPR data indicated that there may be more than one site for CN binding but that one of these may be the tetrahedral iron atom that binds cysteine in the protein. 

Strong evidence to support FeMoco as the site of substrate binding and reduction comes from the study of nif mutants. (The V designation is somewhat unfortunate, as nifV has nothing to do with vanadium.) The NifV mutants do not fix nitrogen in vivo, and have altered substrate specificity in... 

Recently, site-directed mutagenesis studieshave shown that cysteine residues are involved in binding FeMoco to the subunits of [FeMo]. Moreover, these studies again implicate FeMoco in the substrate-reducing site. 

However, this model does not explain why, in the comparable experiment performed under HD, no D2 forms, nor why substrates other than N2 do not promote HD formation. Also, if H2 can interact with the active site, why is a substrate of any kind needed to promote HD formation Displacement of H2 is not a necessity for binding N2, but why does HD form only when N2 is being reduced One simple answer proposed by Helleren et al. is that HD formation and N2 binding occur at different places.54 It is possible that different substrates bind to and are transformed at different parts of the large FeMoco (FeMo cofactor) site of N-ases discussed below. CO inhibits nitrogen fixation in N-ases but not H2 evolution. A single site that binds H2 and N2 equivalently should be poisoned by CO for both H2 and N2 activity, and evidence increasingly points to multisite processes in the FeMoco cluster. However, a possible model (10) for HD formation at the same site as N2 activation is discussed below. 

When FeMoco extracted from MoFe protein purified from a nifV mutant is recombined with apo-MoFe protein, the activated protein has the substrate-reducing characteristics of the nifV enzyme (reduces C2H2 effectively but N2 only poorly). This observation provides the most compelling evidence that FeMoco is, or forms part of, the active site of nitrogenase. Site-directed mutagenesis has implicated one of the conserved Cys residues of the a subunit Cys 275 in binding FeMoco, and also His 196 and Gin 192 (see Refs. 17 and 38 for discussion). 

The iron-molybdenum cofactor ( FeMoco or M cluster ) is widely accepted to be the site of N2 binding and reduction. Mutations near this site, or the absence of homocitrate (from nifV mutants) greatly affect the catalytic specificity and activity. The enzyme is inactive if the biosynthetic machinery for FeMoco synthesis is removed, but becomes active if extracted FeMoco (see Section 8.22.2.3.4) is added. The characteristic EPR signal for the FeMoco changes under turnover conditions, and, as shown below, some substrates have been shown to bind at the FeMoco. 

Nitrogenase, as must now become clear, is a complex enzyme of two component proteins which requires ATP, a reductant, a reducible substrate, Mg " " as an activator, and an anaerobic environment to function. To this complexity must be added the difficulty that the component proteins have no enzymatic half reactions . There are, perhaps, four main questions to decide about the mechanism (1) the role(s) of the two component proteins (2) the role(s) of ATP (3) the nature of the active site(s) and (4) the mechanism of N2 reduction. Despite the complexities and difficulties mentioned above, progress in the last 15 years has partly answered all these questions. The Fe protein mediates an ATP-dependent electron transfer from the donor to the MoFe protein which contains the active site. MgATP binds and induces a conformational change in the Fe protein which lowers its redox potential. FeMoco, the molybdenum cofactor, which may be part of the active site of N2 reduction, has been isolated and partly characterized, while an intermediate in N2 reduction has recently been discovered (Thorneley ct al., 1978). The next part of this chapter describes the evidence for these claims. This evidence involves the noncatalytic reactions of the individual proteins, their... 

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