l-Malate Oxidation by the Electron Transport Fraction of Azotobacter vinelandii

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RESUMO

The membrane-bound l-malate oxidoreductase of Azotobacter vinelandii strain O was found to be a flavoprotein-dependent enzyme associated with the electron transport system (R3) of this organism. The particulate R3 fraction, which possessed the l-malate oxidoreductase, carried out the cyanide-sensitive oxidation of l-malate, d-lactate, reduced nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, succinate, cytochrome c, tetramethyl-p-phenylenediamine, and p-phenylenediamine, with molecular O2 as the terminal electron acceptor. d-Malate was not oxidized, but l-malate was oxidized to oxalacetate. Phenazine methosulfate (PMS), vitamin K3, K3Fe(CN)6, nitro blue tetrazolium, and dichloroindophenol all served as good terminal electron acceptors for the l-malate oxidoreductase. Cytochrome c was a poor electron acceptor. Extensive studies on the l-malate oxidase and PMS and K3 reductases revealed that all were stimulated specifically by flavine adenine dinucleotide and nonspecifically by di- or trivalent cations, i.e., Ca++, Ba++, Mn++, Mg++, Fe+++, Ni++, and Al+++. All these activities were markedly sensitive to ethylenediaminetetraacetate (EDTA). The Vmax values for the l-malate oxidase, PMS, and vitamin K3 reductases were, respectively, 3.4, 15.1, and 45.5 μmoles of substrate oxidized per min per mg of protein at 37 C. Spectral studies revealed that the Azotobacter R3 flavoprotein and cytochromes (a2, a1, b1, c4, and c5) were reduced by l-malate. l-Malate oxidase activity was sensitive to various inhibitors of the electron transport system, namely, p-chloromercuriphenylsulfonic acid, chlorpromazine, 2-n-heptyl-4-hydroxyquinoline-N-oxide, antimycin A, and KCN. Minor inhibitory effects were noted with the inhibitors 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione, rotenone, and Amytal.

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