Purification and characterization of NADH oxidase from membranes of Acholeplasma laidlawii, a copper-containing iron-sulfur flavoprotein.
1. NADH oxidase was extracted from the membranes of Acholeplasma laidlawii with buffer containing 3% Triton X-100 and subsequently purified by several chromatographic steps. The final preparation was essentially homogeneous as judged by gel electrophoresis under nondenaturing conditions. 2. The enzyme appears to be a copper-containing iron-sulfur flavoprotein (FMN:CU:Fe:labile S = 1:1:6:6). The enzyme, containing a high fraction of hydrophobic amino acids, is composed of three subunits of molecular weight 65 000, 40 000 and 19 000. 3. When oxygen is used as electron acceptor the purified enzyme demonstrates a specific activity of 58.0 IU/mg of protein and catalyzes the formation of H2O2 in nearly stoichiometric amount. The apparent Km value for NADH is estimated to be 0.4 mM (pH 7.4). NADPH cannot serve as a substrate for the enzyme. In addition to the NADH oxidase activity, the enzyme is able to catalyze electron transfer from NADH to various other electron acceptors (ferricyanide, dichloroindophenol, cytochrome c). Metal-chelating agents and mercurials are shown to inhibit the activity of the enzyme. 4. From electron paramagnetic resonance and optical absorption measurements evidence was obtained that the flavin semiquinone radical in the NADH oxidase has a high air-stability, and that the flavin shuttles between the fully reduced and the semiquinone state upon electron transport from NADH to the electron acceptors. Inhibition of the NADH oxidoreductase activities by superoxide dismutase indicates that O-2 serves as an intermediate in the electron transfer from NADH to all electron acceptors used in this work. In addition to electron transfer via the superoxide radical O-2, an alternative pathway probably involving Fe-S centers is operative. From these results and literature data we present a reaction scheme for electron transport from NADH to the various electron acceptors.
D000128:Acholeplasma laidlawii / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D003300:Copper; D004579:Electron Transport; D005420:Flavoproteins / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*
D005420:Flavoproteins; D007506:Iron-Sulfur Proteins; D009097:Multienzyme Complexes; D003300:Copper; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases