Steady-state kinetic studies of dithionite utilization, component protein interaction, and the formation of an oxidized iron protein intermediate during Azotobacter vinelandii nitrogenase catalysis.
Steady-state kinetic analysis of the two-component protein system of Azotobacter vinelandii (Av) nitrogenase is reported. A precisely obeyed half-order reaction in dithionite was observed at concentrations up to 21 mM with no indication of saturation by this substrate. This behavior was monitored by optical, amperometric, and manometric kinetic techniques, and the results were mathematically fit to establish the half-order reaction in dithionite. Under conditions where the MgATP and dithionite concentrations remain unchanged, Av2 (the Fe protein component) interacts with Av1 (the MoFe protein component according to the rate law, suggesting a rapid 1:1 Av2-Av1 interaction: [formula: see text]. with [Av2] the free Fe protein concentration, K = 5.9 microM, and Vmax = 2314 nmol of H2 min-1 (mg of Av1)-1. Under dithionite-depleted conditions, Av2 undergoes an Av1-mediated, one-electron oxidation, consistent with its proposed role as a specific, single-electron reductant for Av1. During steady-state turnover as a function of Av2/Av1 ratio, optical spectroscopy demonstrated the presence of 25-30% oxidized Av2 as an enzyme intermediate. Computer-averaged EPR spectra showed that Av1 was > 95% EPR-silent and Av2 was up to 30% oxidized (Av2ox), consistent with the optical measurements. These optical and EPR results show that up to six Av2ox per Av1 can accumulate in the presence of dithionite during catalysis, suggesting that the conversion of Av2ox back into Av2red is a relatively slow process.
Johnson
J L
JL
Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, USA.
Tolley
A M
AM
Erickson
J A
JA
Watt
G D
GD
D016948:Azotobacter vinelandii / Q000201:enzymology*; D004227:Dithionite / Q000378:metabolism*; D006859:Hydrogen / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism; D009591:Nitrogenase / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D013053:Spectrophotometry
D008667:Metalloproteins; D004227:Dithionite; D006859:Hydrogen; D007501:Iron; D009591:Nitrogenase