Oxidation of d (−) Lactate by the Electron Transport Fraction of Azotobacter vinelandii

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Abstract
D(−) Lactate oxidation in Azobacter vinelandii strain O is readily carried out by the membrane bound enzyme that concentrates in the electron transport fraction (R3). This oxidation in the R3 fraction is not dependent on externally added nicotinamide adenine dinucleotide, flavine adenine dinucleotide, or flavine mononucleotide. Phenazine methosulfate, O2, and menadione all served as good electron carriers, and the oxidation of lactate was limited to the d(−) stereoisomer. Of all the α-hydroxyacids examined, only d(−) lactate and d(−) α-hydroxybutyrate were oxidized by the R3 fraction. Paper chromatographic studies revealed that the 2,4-dinitrophenylhydrazine derivative formed from d(−) lactate oxidation was pyruvate. Pyruvate, in turn, could be further decarboxylated nonoxidatively by the R3 fraction. Spectral studies revealed that both the R3 flavoprotein and cytochrome (a2, a1, b1, c4, and c5) components were reduced by d(−) lactate. The d(−) lactic oxidase activity was sensitive to electron transport inhibitors, i.e., chlorpromazine, antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide, rotenone, dicumarol, and cyanide, and to a small extent to 4,4,4-trifluoro-1-(2-thienyl)-1,3-butane-dione (TFTB) and Amytal. The d(−) lactic phenazine methosulfate and menadione reductases were sensitive only to dicumarol and TFTB. Chlorpromazine was found to be a highly specific inhibitor of d(−) lactic oxidase activity, 50% inhibition occurring at 6.6 × 10−6m.

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