Nitrogenase of Azotobacter chroococcum. Kinetics of the reduction of oxidized iron-protein by sodium dithionite

Abstract

The kinetics of the reduction of oxidized Fe-protein of nitrogenase from A. chroococcum by sodium dithionite were studied by stopped-flow and rapid-freezing EPR spectroscopy. The appearance of the gav. = 1.94 EPR signal (0.24 electron integrated intensity/mol) was associated with a 1-electron reduction by SO2- with k > 108 M-1 .cntdot. s-1 at 23.degree. C. A value of k = 1.75 s-1 was obtained for the rate of dissociation of S2O42- into 2SO2- at 23.degree. C. Further reductions by SO2- occurred in three slower phases with rate constants in the range 104-106 M-1 .cntdot. s-1. These latter phases have no corresponding EPR signal changes and are probably associated with enzymically inactive protein. The high rate of reduction by SO2- of the Fe-protein alone (k > 108 M-1 .cntdot. s-1) relative to the rate of oxidation of the Fe-protein in the catalytically active Fe:Mo-Fe protein complex (k = 2.2 .times. 102 s-1) and the observation that in the steady state the Fe-protein is substantially oxidized means that at normal assay concentrations another reaction must limit the rate of reduction of Fe-protein during turnover.