Journal
BIOCHEMISTRY
Volume 40, Issue 6, Pages 1540-1549Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi0013997
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- NIGMS NIH HHS [R01-GM59087] Funding Source: Medline
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EPR signals observed under CO and C2H2 during nitrogenase turnover were investigated for the alpha -Gln(195) MoFe protein, an altered form for which the alpha -His(195) 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 wildtype 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 (S-EPR1) originating from the FeMo-cofactor having two or more bound C2H2 adducts and a second signal (S-EPR2) 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-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 S-EPR2 suggests that it either represents an unusual metal cluster or is a carboxylate centered radical possibly originating from homocitrate. 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 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 alpha -Gln(195) was crystallographically determined and found to be virtually identical to that of the wild-type MoFe protein except for replacement of an NH-S hydrogen bond interaction between FeMo-cofactor and the imidazole side chain of alpha -His(195) by an analogous interaction involving Gin.
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