Nitrogenase reactivity: cyanide as substrate and inhibitor.

Abstract

We have examined the reduction of cyanide by using the purified component proteins of nitrogenase (Av1 and Av2). The previously reported self-inhibition phenomenon was found to be an artifact. One of the two species present in cyanide solutions, CN-, was shown to be a potent reversible inhibitor (Ki = 27 microM) of total electron flow, apparently uncoupling MgATP hydrolysis and electron transfer. There appears to be no differential effect of CN- on the specific activities of Av1 and Av2 nor is there any apparent irreversible physical damage to Av2. CN- inhibition is completely reversed by low levels of CO, implying a common binding site. Azide partially relieves the inhibitory effect, but other substrates and inhibitors (N2, C2H2, N2O, H2) have no effect. The other species present in cyanide solutions, HCN, was shown to be the substrate (Km = 4.5 mM at Av2/Av1 = 8), and extrapolation of the data indicates that at high enough HCN concentration H2 evolution can be eliminated. The products are methane plus ammonia (six electrons), and methylamine (four electrons). There is an excess (relative to methane) of ammonia formed, which, according to electron balance studies, may arise from a two-electron intermediate. Both nitrous oxide and acetylene (but not N2) influence the distribution of cyanide reduction products, implying simultaneous binding. HCN appears to bind to and be reduced at an enzyme state more oxidized than the one responsible for either H2 evolution or N2 reduction.