MOLYBDENUM COFACTOR PROPERTIES AND [FE-S] CLUSTER COORDINATION IN ESCHERICHIA-COLI NITRATE REDUCTASE-A - INVESTIGATION BY SITE-DIRECTED MUTAGENESIS OF THE CONSERVED HIS-50 RESIDUE IN THE NARG SUBUNIT

Most Of the molybdoenzymes contain, in the amino-terminal region of th eir catalytic subunits, a conserved Cys group that in some cases binds an [Fe-S] cluster. In dissimilatory nitrate reductases, the first Cys residue of this motif is replaced by a conserved His residue. Site-di rected mutagenesis of this residue (His-50) was performed on the NarG subunit from Escherichia coli nitrate reductase A. The results obtaine d by EPR spectroscopy enable us to exclude the implication of this res idue in [Fe-S] binding. Additionally, we showed that the His-50 residu e does not coordinate the molybdenum atom, but its substitution by Cys or Ser introduces a perturbation of the hydrogen bonding network arou nd the molybdenum cofactor. From potentiometric studies, it is propose d that the high-pH and the low-pH forms of the Mo(V) are both involved during the redox turnover of the enzyme. Perturbation of the Mo(V) pK (v) value might be responsible for the low activity reported in the Hi s-50-Cys mutant enzyme. A catalytic model is proposed in which the pro tonation/deprotonation of the Mo(V) species is an essential step. Thus , one of the two protons involved in the catalytic cycle could be the one coupled to the molybdenum atom in the dissimilatory nitrate reduct ase of E. coli.