Role of the MoFe Protein .alpha.-Subunit Histidine-195 Residue in FeMo-cofactor Binding and Nitrogenase Catalysis

Abstract

Site-directed mutagenesis and gene-replacement procedures were used to isolate mutant strains of Azotobacter vinelandii that produce altered MoFe proteins in which the alpha-subunit residue-195 position, normally occupied by a histidine residue, was individually substituted by a variety of other amino acids. Structural studies have revealed that this histidine residue is associated with the FeMo-cofactor binding domain and probably provides an NH-->S hydrogen bond to a central bridging sulfide located within FeMo-cofactor. Substitution by a glutamine residue results in an altered MoFe protein that binds but does not reduce N2, the physiological substrate. Although N2 is not a substrate for the altered MoFe protein, it is a potent inhibitor of both acetylene and proton reduction, both of which are otherwise effectively reduced by the altered MoFe protein. This result provides evidence that N2 inhibits proton and acetylene reduction by simple occupancy of a common active site. N2 also uncouples MgATP from proton reduction catalyzed by the altered MoFe protein but does so without lowering the overall rate of MgATP hydrolysis. Thus, the quasi-unidirectional flow of electrons from the Fe protein to the MoFe protein that occurs during nitrogenase turnover is controlled, in part, by the substrate serving as an effective electron sink. Substitution of the alpha-histidine-195 residue by glutamine also imparts to the altered MoFe protein hypersensitivity of both its acetylene reduction and N2 binding to inhibition by CO, indicating that the imidazole group of the alpha-histidine-195 residue might protect an Fe contained within the FeMo-cofactor from attack by CO. Finally, comparisons of the catalytic and spectroscopic properties of altered MoFe proteins produced by various mutant strains suggest that the alpha-histidine-195 residue has a structural role, which serves to keep FeMo-cofactor attached to the MoFe protein and to correctly position FeMo-cofactor within the polypeptide matrix, such that N2 binding is accommodated.

Isolation and Characterization of an Acetylene-resistant Nitrogenase

“…Assays-The composition of the assay mixture and overall techniques used are described elsewhere (11,28). For each assay, 0.05 mg of MoFe protein was used, whereas the Fe protein quantity was adjusted to give the component protein molar ratio shown in Table II.…”

“…Ethylene production was monitored using a HewlettPackard 5890A gas chromatograph equipped with an Al 2 O 3 capillary column and a flame ionization detector. Ammonia production was monitored using the colorimetric indophenol assay previously described (11,30). To reduce background contamination, glass vials used for NH 3 determination were soaked overnight in 1 M NaOH, rinsed thoroughly with distilled water and then ethanol, and oven-dried.…”

“…3). Substitution of ␣-His 195 by glutamine results in an altered MoFe protein that can bind nitrogen with normal affinity but does not significantly reduce it (11). Nevertheless, the ␣-Gln 195 MoFe protein is unaltered in its ability to either bind or reduce acetylene, so the high affinity acetylene-binding/reduction site must remain intact.…”

“…Of particular interest to our laboratories is the contribution of the FeMo cofactor polypeptide environment toward its ability to bind and effect the reduction of various substrates. In previous work we gained insight about the functioning of nitrogenase by characterizing an altered Azotobacter vinelandii MoFe protein for which the ␣-subunit His 195 residue was substituted by glutamine (11). This altered MoFe protein does not significantly reduce nitrogen (11) but is still able to reduce acetylene and does so with essentially unaltered kinetic parameters.…”

Diazene (HNNH) Is a Substrate for Nitrogenase: Insights into the Pathway of N2 Reduction

“…However, based on its g values, this appears to be from an N 2 -turnover state, probably formed with N 2 generated as a breakdown product of diazene (49). In contrast, conversion to an intermediate with a distinct EPR signal is observed when the α-195 Gln MoFe protein is trapped during turnover with diazene; the substitution of α-195 His by Gln has been suggested to limit proton delivery for reduction of nitrogenous substrates, and thus to arrest the reduction of these substrates (36,52,53 It was important to establish that the trapped state observed by EPR is a result of diazene binding to or reacting with nitrogenase, and not one of the diazene breakdown products. In an experiment parallel to the a kinetic studies described above, diazene was allowed to decompose for 30 min in an EPR tube prior to the addition of the nitrogenase proteins.…”