Nitrogenase of Klebsiella pneumoniae. Kinetics of the dissociation of oxidized iron protein from molybdenum-iron protein: identification of the rate-limiting step for substrate reduction.
Stopped-flow spectrophotometry and e.p.r. spectroscopy were used to study the kinetics of reduction by dithionite of the oxidized Fe protein of nitrogenase from Klebsiella pneumoniae (Kp2ox.) in the presence of MgADP at 23 degrees C at pH 7.4. The active reductant, SO2.-, produced by the predissociation of S2O4(2-) in equilibrium 2SO2.-, reacts with Kp2ox. (MgADP)2, with k4 = 3.0 X 10(6) +/- 0.4 X 10(6) M-1 X s-1. The inhibition of this reaction by the Mo-Fe protein (Kp1) has enabled the rate of dissociation of Kp2ox. (MgADP)2 from Kp1+ (the Kp2-binding site on Kp1) to be measured (k-3 = 6.4 +/- 0.8 s-1). Comparison with the steady-state rate of substrate reduction shows that the dissociation (k-3) of the complex Kp2ox. (MgADP)2-Kp1+, which is formed after MgATP-induced electron transfer from Kp2 to Kp1+, is the rate-limiting step in the catalytic cycle for substrate reduction.
D000244:Adenosine Diphosphate / Q000378:metabolism; D004227:Dithionite / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D005288:Ferredoxins / Q000378:metabolism*; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D008983:Molybdoferredoxin / Q000378:metabolism*; D009591:Nitrogenase / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases; D013053:Spectrophotometry; D013458:Sulfur Dioxide / Q000378:metabolism
D005288:Ferredoxins; D008983:Molybdoferredoxin; D013458:Sulfur Dioxide; D004227:Dithionite; D000244:Adenosine Diphosphate; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase