Mechanistic Features and Structure of the Nitrogenase α-Gln195 MoFe Protein,

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Abstract
EPR signals observed under CO and C2H2 during nitrogenase turnover were investigated for the α-Gln195 MoFe protein, an altered form for which the α-His195 residue has been substituted by glutamine. Under CO, samples show S = 1/2 hi- and lo-CO EPR signals identical to those recognized for the wild-type protein, whereas the S = 3/2 signals generated under high CO/high flux conditions differ. Previous work has revealed that the EPR spectrum generated under C2H2 exhibits a signal (SEPR1) originating from the FeMo-cofactor having two or more bound C2H2 adducts and a second signal (SEPR2) arising from a radical species [Sørlie, M., Christiansen, J., Dean, D. R., and Hales, B. J. (1999) J. Am. Chem. Soc. 121, 9457−9458]. Pressure-dependent studies show that the intensity of these signals has a sigmoidal dependency at low pressures and maximized at 0.1 atm C2H2 with a subsequent decrease in steady-state intensity at higher pressures. Analogous signals are not recognized for the wild-type MoFe protein. Analysis of the principal g-factors of SEPR2 suggests that it either represents an unusual metal cluster or is a carboxylate centered radical possibly originating from homocitrate. Both SEPR1 and SEPR2 exhibit similar relaxation properties that are atypical for S = 1/2 signals originating from Fe−S clusters or radicals and indicate a coupled relaxation pathway. The α-Gln195 MoFe protein also exhibits these signals when incubated under turnover conditions in the presence of C2H4. Under these conditions, additional inflections in the g 4−6 region assigned to ground-state transitions of an S = 3/2 spin system are also recognized and assigned to turnover states of the MoFe protein without C2H4 bound. The structure of α-Gln195 was crystallographically determined and found to be virtually identical to that of the wild-type MoFe protein except for replacement of an ΝH−S hydrogen bond interaction between FeMo-cofactor and the imidazole side chain of α-His195 by an analogous interaction involving Gln.