Binding Sites of Nitrogenase: Kinetic and Theoretical Studies of Cyanide Binding to Extracted FeMo-Cofactor Derivatives

Abstract

The first kinetic study of a substrate (CN-) binding to the isolated active site (extracted FeMo-cofactor) of nitrogenase is described. The kinetics of the reactions between CN- and various derivatives of extracted FeMo-cofactor {FeMoco−L; where L is bound to Mo, and is NMF, ButNC, or imidazole (ImH)} have been followed using a stopped-flow, sequential-mix method in which the course of the reaction is followed indirectly, by monitoring the change in the rate of the reaction of the cofactor with PhS-. The kinetic results, together with DFT calculations, indicate that the initial site of CN- binding to FeMoco−L is controlled by a combination of the electron-richness of the cluster core and lability of the Mo−L bond. Ultimately, the reactions between FeMoco−L and CN- involve displacement of L and binding of CN- to Mo. These reactions occur with a variety of rates and rate laws dependent on the nature of L. For FeMoco−NMF, the reaction with CN- is complete within the dead-time of the apparatus (ca. 4 ms), while with FeMoco−CNBut the reaction is much slower and exhibits first order dependences on the concentrations of both FeMoco−CNBut and CN- (k = 2.5 ± 0.5 × 104 dm3 mol-1 s-1). The reaction of FeMoco−ImH with CN- occurs at a rate which exhibits a first order dependence on FeMoco−ImH but is independent of the concentration of CN- (k = 50 ± 10 s-1). The results are interpreted in terms of CN- binding directly to the Mo site for FeMoco−NMF and FeMoco−ImH, but with FeMoco−CNBut initial binding at an Fe site is followed by movement of CN- to Mo. Complementary DFT calculations are consistent with this interpretation, indicating that, in FeMoco−L, the Mo−L bond is stronger for L = ImH than for L = CNBut and the binding of CN- to Mo is stronger than to any Fe atom in the cofactor.