Electron-paramagnetic-resonance studies on nitrate reductase from Escherichia coli K12.

Nitrate reductase was purified from anaerobically grown Escherichia coli K12 by a method based on the Triton X-100 extraction procedure of Clegg[(1976) Biochem. J.153, 533-541], but hydrophobic interaction chromatography was used in the final stage. E.p.r. spectra obtained from the enzyme under a variety of conditions are well resolved and were interpreted with the help of the computer-simulation procedures of Lowe [(1978) Biochem. J.171, 649-651]. Parameters for five molybdenum(V) species from the enzyme are given. The low-pH species (g(av.) 1.9827) is in pH-dependent equilibrium with the high-pH species (g(av.) 1.9762), the pK for interconversion of the species being 8.26. Of a variety of anions tested, only nitrate and nitrite formed complexes with the enzyme (in the low-pH form), giving modified molybdenum(V) e.p.r. spectra. These complexes, as well as the low-pH form of the free enzyme, showed interaction of molybdenum with a single exchangeable proton. The fifth molybdenum(V) species, sometimes detected in small amounts, appears not to be due to functional nitrate reductase. After full reduction of the enzyme with dithionite, addition of nitrate caused reoxidation of molybdenum to the quinquivalent state, in a time less than the enzyme turnover. Activity of the enzyme in the pH range 6-10 is controlled by a pK of 8.2. It is suggested that the low-pH signal-giving species is the form of the enzyme involved in the catalytic cycle. Iron-sulphur and other e.p.r. signals from the enzyme are briefly described and the enzymic reaction mechanism is discussed.