FeMoco substrate binding

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. 

The second example is the measurement of the kinetics of a substrate binding to extracted FeMo-cofactor of nitrogenase by a double-mixing stopped-flow approach. In a typical experiment, solutions of FeMoco-L and CN are rapidly mixed and held together for a known length of time. Subsequently, this solution is mixed with a solution of [NEt4]SPh whereupon the thiolate reacts with the cofactor. 

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... 

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. 

In general there are few reproducible data on binding of reducible substrates to the isolated MoFe proteins. However, the S = EPR signal from the FeMoco centers of Kpl is pH dependent, the g values changing with a pKa of 8.7 (50). Of course, the proton is a substrate of nitrogenase however, there is no direct evidence for the proton associated with the pKa being bound directly to FeMoco. Nevertheless, this pKa can be perturbed by addition of the analog substrate acety-... 

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). 

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. 

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. 

Extracted FeMoco is not active for the reduction of N2. However, it has been observed to reduce protons to H2 electrochemically. It also reacts with alternative substrates (Section 8.22.3.3) for example, it binds multiple molecules of cyanide. It will catalyze the reduction of acetylene to ethylene and ethane by metal amalgam reducing agents. In an interesting observation, N2 is a competitive inhibitor of acetylene reduction, providing the first evidence for N2 binding at extracted FeMoco. ... 

---