Purification and Properties of NADH Oxidase from Bacillus megaterium1

Abstract

NADH oxidase, which catalyzes the oxidation of NADH, with the consumption of a stoichiometric amount of oxygen, to NAD⁺ and hydrogen peroxide was purified from Bacillus megaterium by 5′-AMP Sepharose affinity chromatography to homogeneity. The enzyme is a dimeric protein containing 1 mol of FAD per mol of subunit, Mr=52,000. The absorption maxima of the native enzyme (oxidized form) were found at 270, 383, and 450 with a shoulder at 475 nm in 50 mM KP₁ buffer, pH 7.0. The visible absorption bands at 383 and 450 nm disappeared on the addition of NADH under anaerobic conditions and reappeared upon the introduction of air. Thus, the non-covalently bound FAD functioned as a prosthetic group for the enzyme. We tentatively named this new enzyme NADH oxidase (NADH:oxygen oxidoreductase, hydrogen peroxide forming). This enzyme stereospecifically oxidizes the pro-S hydrogen at C-4 of the pyridine ring of NADH.