title: 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.
abstract: The pre-steady-state kinetics of H2 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 H2 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 (kobs. = 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.
authors.affiliation: AFRC Institute of Plant Science Research, Nitrogen Fixation Laboratory, University of Sussex, Brighton, U.K.
authors.forename: K
authors.initials: K
authors.lastname: Fisher
authors.forename: D J
authors.initials: DJ
authors.lastname: Lowe
authors.forename: R N
authors.initials: RN
authors.lastname: Thorneley
mesh_terms: D003198:Computer Simulation; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D006859:Hydrogen / Q000378:metabolism*; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D008983:Molybdoferredoxin / Q000378:metabolism*; D009591:Nitrogenase / Q000378:metabolism*; D010084:Oxidation-Reduction; D013057:Spectrum Analysis
chemical_list: D008983:Molybdoferredoxin; D006859:Hydrogen; D009591:Nitrogenase