We have examined the interaction of HCN and CN" with wild type and two altered Azotobacter vinelandii nitrogenase MoFe proteins containing substitutions at the a- 195His position (al95Gln and al95Asn). This residue is of considerable interest because previous work has shown that the a 195 n altered MoFe protein is ineffective in normal N2 and azide reduction but effective in HCN and H+ reduction even though the hydrogen bond to the central sulfide of the FeMo-cofactor remains intact (Dilworth et al. 1998). We have determined the relative rates of CH4, NH3, CH3-NH2 and H2 product formation from HCN-reduction assays as a function of HCN concentration. It was necessary to separate the ammonia and methylamine before product determination because the phenol-hypochlorite method commonly used to measure ammonia also recovers methylamine. In particular, we have focused on the source of the "excess ammonia". Unlike the wild type and al95Asn MoFe proteins, the al95Gln MoFe protein catalyzes HCN reduction without the production of "excess ammonia" and without suffering significant inhibition by CN". We propose that the lack of production of "excess ammonia" is a consequence of a substantially lower affinity of the al95Gln MoFe protein for HCN binding, i.e. HCN is less able to displace intermediates from the active site. We have developed a sensitive assay, using acetylacetone, to determine if formaldehyde is a product of HCN reduction. We were able to accurately recover (>95%) formaldehyde (10-50 nmol) from supplemented mock nitrogenase assays. However, nitrogenase assays conducted with either wild type or al95Asn MoFe protein in the presence of 1 mM NaCN, emphatically showed that formaldehyde was not produced. Our search for the missing product continues.
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