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

Abstract

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 (R 3 ) of this organism. The particulate R 3 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 O 2 as the terminal electron acceptor. d -Malate was not oxidized, but l -malate was oxidized to oxalacetate. Phenazine methosulfate (PMS), vitamin K 3 , K 3 Fe(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 K 3 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 V max values for the l -malate oxidase, PMS, and vitamin K 3 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 R 3 flavoprotein and cytochromes ( a 2 , a 1 , b 1 , c 4 , and c 5 ) 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.