At neutral pH in nitrogenase assays, cyanide is present as the substrate HCN (-98%) and the potent reversible inhibitor CN" (-2%). HCN is reduced by six electrons to CH4 and NH3j and four electrons to CH3-NH2. Under certain conditions, additional ammonia can also be formed during nitrogenase-catalyzed HCN reduction. "Excess ammonia", i.e. the amount of NH3 in excess of CH4, has been suggested to arise from an initial two-electron/two-proton step converting HCN into CH2=NH (methyleneimine). When any of this intermediate escapes from the active site, it is hydrolyzed to formaldehyde, which has never been detected, and "excess ammonia". Additional two-electron/two-proton steps yield CH3-NH2, some of which escapes, and finally CH4 and NH3 (Li et al. 1982).
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