Les nitrate‐réductases bactériennes

Solubilization and Purification. Crude extract is perpared from cells of Micrococcus denitrificans grown anaerobically in the presence of NO3−. Nitrate reductase A is solubilized by an alkaliacetone treatment of particles. The purification consists of four steps: ammonium sulfate precipitation at 35% saturation; Sephadex gel filtration; discontinuous elution from two successive phosphate gel columns, the first eluted by acid phophate bffer, the second by alakline. The enzyme is purified 50-fold with a yield of 17%. Polyacrylamide gel electrophoresis and ultracentrifugation show the prepartion to be nearly homogeneous (95% purity). Properties of the Purified Enzyme. Isoelectric point: pH 4.2. Molecular weight: (filtration on Sephadex and ultracentrifugation) 160000. Presence of flavins: no FMN and FAD. Absorption spectra: purified preparations have a brown color. Absorption increases continuously from 550 to 280 nm but does not exhibit any peaks in the visible or near-ultraviolet. In the visible, the higher the specific absorption the higher the iron content of the protein. Heavy metals: the enzyme contains iron but the content varies from one preparation to another, 4 to 8 atoms per mole (Mw= 160000). Some of the iron is lost during purification and 8 atoms sees the value closer to reality. Traces of molybdenum are revealed by analysis, but may be acontamination although we cannot discard the hypothesis that this metal is normally present in the enzyme and lost during the purification. In ths case, it must be assumed that molybdenum is not necessary for the activity of nirate reductase with the artificaial electrons donors used. Labile sulfide: approximately one labile sulfide is found per iron atom. Amino acid composition: high content in aspartic and glutamic acids; low content in cysteine residues. Electron donors: the enzyme uses the reduced forms of viologen indicators, FMNH2, FADH2 but not NADH and NADPH. Substrates: NO3−, C1O3− and BrO3− are substrates. Inhibitors: CN− and N3− are inhibitors. The observed kinetics with 33.3 and 3.33 μM N3− are those of competitive inhibition. The nitrate reductase affinity for azide is one thousand times greater than for nitrate or chlorate. Versene and 1, 10-phenanthroline are not inhibitors. Influence of pH: activity is maximal at pH 5.6-5.8 with NO3− and at 6.2–6.4 with C1O3−. Iron function: the following observations suggest this metal plays an essential role in nitrate reductase A catalysis: (a) the higher the iron content of the purified fraction the greater its activity; (b) the 2 inhibitors, CN− and N3−, complex heavy metals; (c) dithionite causes a lowering of the absorption between 300 and 500 nm and this effect is reversed by the addition of nitrate; (d) the competitive inhibition by azide indicates that the iron may be at the active center and may play an important role in the formation of the enzyme-substrate complex. Conclusion. Nitrate reductase A of M. denitrificans is a non-heme iron protein.