Nitrogenase MoFe protein mutants

An Fe-only nitrogenase has also been isolated from a nifH mutant of Rhodospirillum rubrum and was characterized as an a2/82<% hex-amer containing only iron, no molybdenum or vanadium, with an o 2Fe4S4-containing Fe protein. A factor could be extracted from the FeFe protein into NMF that combined with apo-MoFe protein to form an active enzyme 193). 

Inactive MoFe protein may also be isolated from the nitrogenase of nif B mutants of K. pneumoniae.14 4 Addition of FeMoco results in activation up to 40% of normal activity, apparently because only one mol of FeMoco could be added. The nif B preparation appeared to contain one atom of non-FeMoco Mo, and so formed the MoFe protein with two Mo but only one FeMoco. This suggests that the two FeMoco sites in wild-type MoFe protein operate independently.1485 The schematic representation of nitrogenase shown in Figure 72 thus contains two peptides, two P clusters and one FeMoco group. 

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). 

Cyanide and nitriles are reduced to alkanes and ammonia with nitrogenase and reductant. (Cyanide is in equilibrium with HCN in aqueous solution.) Cyanide binds to the S = 3/2 state of the extracted FeMoco in solution, but not to the M state of holo-MoFe protein. Cyanide binds to mutants with changes in the belt region, as is evident from C ENDOR signatures. Kinetics data suggest that cyanide binds to a more oxidized state of the FeMoco than N2 does, and a lag time may indicate a rearrangement. ... 

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