Mechanistic features and structure of the nitrogenase alpha-Gln(195) MoFe protein

EPR signals observed under CO and C2H2 during nitrogenase turnover were inv estigated for the alpha -Gln(195) MoFe protein, an altered form for which t he alpha -His(195) residue has been substituted by glutamine. Under CO, sam ples show S = 1/2 hi- and lo-CO EPR signals identical to those recognized f or the wildtype protein, whereas the S = 3/2 signals generated under high C O/high flux conditions differ. Previous work has revealed that the EPR spec trum generated under C2H2 exhibits a signal (S-EPR1) originating from the F eMo-cofactor having two or more bound C2H2 adducts and a second signal (S-E PR2) arising from a radical species [Sorlie, M., Christiansen, J., Dean, D. R., and Hales, B. J. (1999) J Am. Chem. Sec. 121, 9457-9458]. Pressure-dep endent studies show that the intensity of these signals has a sigmoidal dep endency at low pressures and maximized at 0.1 atm C2H2 With a subsequent de crease 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 S-EPR2 suggests that it either represents an unusual metal clus ter or is a carboxylate centered radical possibly originating from homocitr ate. Both S-EPR1 and S-EPR2 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 alpha -Gln(195) MoFe protein al so exhibits these signals when incubated under turnover conditions in the p resence 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 alpha -Gln(195) was crystallographically dete rmined and found to be virtually identical to that of the wild-type MoFe pr otein except for replacement of an NH-S hydrogen bond interaction between F eMo-cofactor and the imidazole side chain of alpha -His(195) by an analogou s interaction involving Gin.