Cyanide and methylisocyanide: Probes for nitrogenase reactivity

Abstract Nitrogenase (N 2 ase) is composed of two separately purified proteins, the molybdenum-iron (MoFe) protein and the iron (Fe) protein. Nitrogen fixation requires both proteins, a reductant, protons and MgATP. The Fe protein is generally accepted as a specific one-electron donor for the MoFe protein, which is believed to contain the substrate-reduction site. Besides, N 2 , N 2 ase catalyzes the reduction of protons and a number of alternative substrates [ e.g. 1], including the two six-electron substrates, cyanide and methylisocyanide, we have recently studied. The rate-limiting step for N 2 ase turnover occurs prior to substrate reduction. Thus, total electron flow through the enzyme should be essentially independent of the substrate being reduced. Although this appears true N 2 fixation, H 2 evolution and C 2 H 2 reduction [2], both CN − [3] and CH 3 NC dramatically inhibit the rate of total electron flow through N 2 ase. Inhibition by both substrates is completely reversed by CO. Not only do CN− and CH 3 NC inhibit nitrogenase turnover, they also reduce the enzyme's efficiency by increasing the amount of MgATP hydrolyzed for each electron pair used to reduce substrate. These data are interpreted in terms of CN− or CH 3 NC binding to the MoFE protein in such a way as to prevent electron transfer to substrate. With nowhere to go, the electrons fall back to the Fe protein to complete a futile cycle. Are the substrates N 2 , HCN and CH 3 NC reduced in one six-electron step or via a series of lesser reduced intermediates? Previously, we proposed that N 2 is reduced to ammonia via the two-electron intermediates N 2 H 2 and N 2 H 4 [4]. For the six-electron reductions of HCN to CH 4 + NH 3 and CH 3 NC to CH 4 + CH 3 NH 2 , we have definitely identified the four-electron products, CH 3 NH 2 (for HCN) and CH 3 NHCH 3 (for CH 3 NC), and suggest them as intermediates. The formation of two-electron reduced intermediates for both HCN and CH 3 NC is suggested by the product ratio of NH 3 -to-CH 4 (for HCN) and CH 3 NHCH 2 -to-CH 4 (for CH 3 NC) being greater than one. The data support mechanisms whereby the six-electron reduction of N 2 , HCN and CH 3 NC occur via a series of analogous two- and four-electron reduced intermediates. Thus, a common phenomenon is likely as an intimate part of the mechanisms of N 2 , HCN and CH 3 NC reduction. Although H 2 evolution is suggested as an obligatory part of the N 2 -fixation mechanism, it is not required for either HCN or CH 3 NC reduction. This apparent anomaly might be explained if N 2 , HCN or CH 3 NC were either reduced at different sites or bound and reduced by different redox states of the MoFe protein. So, as increasing the ratio of Fe protein-to-MoFe protein increases electron flow, component protein ratio titration experiments in the presence of N 2 ,HCN and CH 3 NC were used. They indicate that HCN and CH 3 NC bind to and are reduced at a redox state of the MoFe protein more oxidized than that responsible for either N 2 fixation or H 2 evolution. Do all substrates and inhibitors of nitrogenase bind to the same site on nitrogenase? Experiments with various combinations of substrates (N 2 , HCN, CH 3 NC, C 2 H 2 , N 2 O, N − 3 and inhibitors (H 2 , CO, CN − , CH 3 NC) indicate that either C 2 H 2 or N 2 O stimulate HCN reduction and influence its product distribution, implying simultaneous binding and at least two interaction sites on N 2 ase. CH 3 NC appears to act as both substrate and inhibitor on binding to the same N 2 ase site, implying productive and non-productive modes of binding.