Les nitrate-reductases bacteriennes solubilisation, purification et proprietes de l'enzyme a de Micrococcus halodenitrificans

Abstract

Solubilization and purification. Crude extract is prepared from cells of Micrococcus halodenitrificans grown anaerobically in the presence of NO 3 −. Nitrate reductase A is solubilized by an alkali-acetone treatment of particles. The purification consists of four steps: Sephadex gel filtration; elimination of nucleic acids by protamine sulfate precipitation; two successive absorptions and elutions on Ca 3(PO 4) 2 gel. The enzyme is purified 40-fold with a yield of 12%. Polyacrylamide gel electrophoresis) show the preparation to be homogeneous. Properties. Molecular weight: (electrophoresis 165 000. Flavins: no FMN or FAD. Purified preparations have a brown color. Absorption increases continuously from 600 to 280 nm. The spectrum has a shoulder at 410 nm, but no peak in the visible or near-ultraviolet. The enzyme contains approximately 2 Fe atoms, 1 Mo atom and 4 “labile sulfide groups in acid medium” per mole ( M w = 165 000). It has a high content in aspartic and glutamic acids. Electron donors: the enzyme uses the reduced forms of viologen indicators, FMNH 2 but not NADH and NADPH. It uses NO 3 − and ClO 3 −1 as substrates. Cyanide and azide inhibit its activity. The inhibition by azide is competitive. The enzyme's affinity for azide is about a thousand times greater than for NO 3 − or ClO 3 −1. Activity is maximal at pH 6.3 (NO 3 −1 and 6.4 (ClO 3 −1). NaCl does not activate the enzyme. Conclusion. Nitrate reductase A of M. halodenitrificans is a non-heme Fe protein containing Mo.