MoFe protein characteristics

The MoFe proteins isolated from all sources are 2/32 tetramers of Mr —220,000. The DNA sequences encoding the a subunit (nifD) and /3 subunit inifK) have been cloned and sequenced from seven organisms. The derived amino acid sequences indicate considerable homology, in particular live invariant Cys residues in the a subunit and three invariant Cys residues in the /3 subunit. As in the case with the Fe proteins, the conserved ferredoxin-like Cys-X-X-Cys spacing characteristic of many Fe-S proteins is not observed (Fig. 1). (25). Comparison of the sequences of the a and /3 subunits shows some homology, and X-ray... 

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). 

In the dye-oxidized EPR-silent state a cluster that was diamagnetic in the dithionite-reduced state became paramagnetic, with MCD and magnetization characteristics assignable to an S = 5/2 spin system with near axial symmetry. These data are very similar, but are not identical with those for the P clusters of MoFe proteins and suggest the presence of analogous clusters in the VFe proteins. 

The P cluster is an FegS cluster that lies at the interface of the a- and /3-subunits of the MoFe protein. It is situated about 15 A from the site at which the Fe protein binds and about the same distance from the FeMoco, suggesting a role in electron transfer. This idea was first supported by a tandem EPR/kinetics study, in which the (small) characteristic spectroscopic change observed corresponds to the rate of FeMoco reduction.More direct evidence comes from the observation in a mutant of a characteristic EPR signal that disappears during turnover and then returns.Therefore, the P cluster is generally viewed as a gateway for electron transfer into the catalytic FeMoco center. The P cluster is observed in several redox states the most common are called P (or P° ), P " ", and P (fully reduced). 

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