Anaerobic Formation of d-Lactate and Partial Purification and Characterization of a Pyruvate Reductase from Chlamydomonas reinhardtii.

d-Lactate accumulation in Chlamydomonas reinhardtii was dependent on anaerobic conditions. As much as 50% of the (14)C after 2 minutes of photosynthetic (14)CO(2) fixation moved into d-lactate from sugar phosphates if the cells became anaerobic for short time periods. No lactate accumulated in the dark until the O(2) concentration decreased to less than 0.1%. Lactate was determined to be of the d-configuration using stereospecific lactate dehydrogenases. d-Lactate produced anaerobically by algae grown on 5% CO(2) was only slowly metabolized aerobically in the light or dark, and in the dark, only a trace of the lactate was excreted.A pyruvate reductase (d-lactate: diphosphopyridine nucleotide oxidoreductase, EC 1.1.1.28) was partially purified 47-fold from Chlamydomonas. Because this enzyme catalyzes an essentially irreversible reaction in the direction of pyruvate reduction, it is considered to be a pyruvate reductase. The reductase activity in extracts of Chlamydomonas was 30 micromoles per hour per milligram chlorophyll. For the partially purified enzyme, the apparent K(m) (pyruvate) was 0.5 millimolar, and the pH optimum was 7.0. Studies with cycloheximide and chloramphenicol indicated that the enzyme was constitutive in aerobic cells. Potassium phosphate stimulated the reductase, and high salt and dithiothreitol were required for stability. The enzyme demonstrated substrate inhibition and was inhibited by ATP. Pyruvate reductase was separated from a hydroxypyruvate reductase by gel filtration chromatography, indicating the presence of separate reductases for these two substrates in Chlamydomonas.