KLEBSIELLA-PNEUMONIAE NITROGENASE - THE PRE-STEADY-STATE KINETICS OF MOFE-PROTEIN REDUCTION AND HYDROGEN EVOLUTION UNDER CONDITIONS OF LIMITING ELECTRON FLUX SHOW THAT THE RATES OF ASSOCIATION WITH THE FE-PROTEIN AND ELECTRON-TRANSFER ARE INDEPENDENT OF THE OXIDATION LEVEL OF THE MOFE-PROTEIN

The pre-steady-state kinetics of H-2 evolution from Klebsiella pneumoniae nitrogenase functioning at 23-degrees-C, pH 7.4, under conditions of extremely low electron flux through the MoFe-protein exhibited a lag phase of several minutes duration. The approach to a steady-state rate of H-2 evolution was accompanied by a 50% decrease in the amplitude of the MoFe-protein e.p.r. signal. These kinetics have been simulated using our published kinetic model for nitrogenase [Lowe & Thorneley (1984) Biochem. J. 224, 877-886], which was developed using data obtained with nitrogenase functioning at high electron fluxes. The e.p.r. data showed that the rate of complex-formation between reduced Fe-protein and the MoFe-protein (k+1 = 5 x 10(7) M-1.s-1) is the same for the resting (E0) and one-electron-reduced (E1H) states of the MoFe-protein. Stopped-flow spectrophotometry also showed that electron transfer from the Fe-protein to the MoFe-protein in states E0 and E1H occurs at the same rate (k(obs.) = 140 s-1). These data support our previous assumption that the rate constants that define the 'Fe-protein cycle' are independent of the level of reduction of the MoFe-protein.