Complexes with halide and other anions of the molybdenum centre of nitrate reductase from Escherichia coli

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Abstract
The interconversion of nitrate reductase from E. coli between low-pH and high-pH Mo(V) EPR signal-giving species was re-investigated. The process cannot be described by a single pK value, since the apparent pK for interconversion is raised by the presence of various anions. The low-pH form of the enzyme exists as a series of complexes with different anion ligands of molybdenum. Each complex has specific and slightly different EPR parameters, but all show strong coupling of Mo(V) to a single proton, exchangeable with the solvent, having A(1H)av, 1.0 to 1.3 mT. Complexes with Cl-, F- [A(19F)av, 0.7 mT], NO3- and NO2- give particularly well-defined spectra. The high-pH form of the enzyme is now shown to bear a coupled proton. Like that in the low-pH species, this proton is exchangeable with the solvent, but the coupling is much weaker, with A(1H)av, 0.3 mT. Contrary to earlier assumptions, the proton detectable by EPR is probably not identical with the proton whose dissociation controls interconversion between the 2 species; the latter proton could be located in the protein rather than on a ligand molybdenum. Treatment of the enzyme with trypsin did not affect its Mo(V) EPR signals.