The mechanism of Klebsiella pneumoniae nitrogenase action. The determination of rate constants required for the simulation of the kinetics of N2 reduction and H2 evolution

Kinetic data for Klebsiella pneumoniae nitrogenase were used to determine the values of nine of the 17 rate constants that define the scheme for nitrogenase action described by Lowe & Thorneley [(1984) Biochem. J. 224, 877-886]. Stopped-flow spectrophotometric monitoring of the MgATP-induced oxidation of the Fe protein (Kp2) by the MoFe protein (Kp1) was used to determine the rates of association (k+1) and dissociation (k-1) of reduced Kp2(MgATP)2 with Kp1. The dependences of the apparent KNm2 on Fe protein/MoFe protein ratio and H2 partial pressure were used to determine the mutual displacement rates of N2 and H2 (k+10, k-10, k+11 and k-11). These data also allowed the rate constants for H2 evolution from progressively more reduced forms of Kp1 to be determined (k+7, k+8 and k+9). A mechanism for N2-dependent catalysis of 1H2H formation from 2H2 that requires H2 to be a competitive inhibitor of N2 reduction is also presented.