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

Abstract

Stopped-flow spectrophotometry and EPR spectroscopy were used to study the kinetics of reduction by dithionite of the oxidized Fe protein of nitrogenase from K. pneumoniae (Kp2ox.) in the presence of MgADP at 23.degree. C at pH 7.4. The active reductant, SO2.-, produced by the predissociation of S2O42 .dblarw. 2SO2.-, reacts with Kp2ox (MgADP)2, with k4 = 3.0 .times. 106 M-1 .cntdot. s-1. The inhibition of this reaction by the Mo-Fe protein (Kp1) enabled the rate of dissociation of Kp2ox. (MgADP)2 from .**GRAPHIC**. (the Kp2-binding site on Kp1) to be measured (k-3 = 6.4 .+-. 0.8s-1). Comparison with the steady-state rate of substrate reduction shows that the dissociation (k-3) of the complex Kp2ox. .**GRAPHIC**. which is formed after MgATP-induced electron transfer from Kp2 to .**GRAPHIC**. is the rate-limiting step in the catalytic cycle for substrate reduction.