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

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 (R 3 ). This oxidation in the R 3 fraction is not dependent on externally added nicotinamide adenine dinucleotide, flavine adenine dinucleotide, or flavine mononucleotide. Phenazine methosulfate, O 2 , 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 R 3 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 R 3 fraction. Spectral studies revealed that both the R 3 flavoprotein and cytochrome ( a 2 , a 1 , b 1 , c 4 , and c 5 ) 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 −6 m .