The Bacterial Nitrate Reductases

Nitrate reductase of Escherichia coli has been solubilized from particle fractions by a double treatment: first an akali-acetone precipitation and then a solubilization in a buffered sodium deoxycholate. The enzyme has been purified 50-fold with a yield of 1 to 2%. Polyacrylamide-gel electrophoresis and ultracentrifugation show the preparation to be nearly homogeneous. The protein has a molecular weight of 320000 and an iso-electric point at pH 4.2. The absorbance which increases continuously from 600 to 280 nm does not reveal the presence of a heme or a flavin group but the spectrum resembles that of some bacterial ferroproteins. The estimation of metals indicates 1.5 atoms Mo and 20 atoms Fe per mole. Approximately one labile sulfide is found per iron atom. It is likely that nitrate reductase A is an iron-sulfur protein containing molybdenum. The purified protein uses as substrates NO3- and CIO3- and as electron donors reduced benzyl- and methyl-viologens, FMNH2 and FADH2 but not NADH or NADPH. It should be pointed out that the solubilization does not modify the enzymatic properties of nitrate reductase. CN- and N3- are strong inhibitors. Azide is a competitive inhibitor and the nitrate reductase affinity for this inhibitor is 1000 times greater than for nitrate. The type of inhibition observed and the metal chelating nature of the inhibitor suggest that a metal, Fe or Mo, or both, play a role in the formation of enzyme-substrate complex.