Binding sites of nitrogenase: Kinetic and theoretical studies of cyanide binding to extracted FeMo-cofactor derivatives

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 {FeMocoL; where L is bound to Mo, and is NMF, (BuNC)-N-t, 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 x 10(4) dm(3) mol(-1) s(-1)). The reaction of FeMoco-lmH with CN- occurs at a rate which exhibits a first order dependence on FeMoco-lmH 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-lmH, 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 = lmH than for L = CNBut and the binding of CN- to Mo is stronger than to any Fe atom in the cofactor.