Characterization of NADPH: Protochlorophyllide oxidoreductase in the y-7 and pc-1 y-7 mutants of Chlamydomonas reinhardtii

Abstract

Protochlorophyllide (Pchilide) photoconversion, a key step in the biosynthesis of chlorophyll, is catalyzed by the enzyme NADPH: Pchlide oxidoreductase. We have previously shown that the pc-1 mutation of Chlamydomonas reinhardtii causes a defect in photoconversion in vivo. The double mutant pc-1 y-7 failed to photoconvert Pchlide to chlorophyllide, whereas the yellow mutant y-7 showed normal photoconversion (Ford et al. 1981). We have now obtained similar results in vitro, using detergent solubilized membrane preparations. The enzyme activity in y-7 preparations had a pH optimum of 8.5 and had a stringent requirement for both NADPH and Pchlide. We have partially purified the enzyme activity from y-7 by gel filtration. Photoconversion activity eluted with a 320,000 dalton membrane-protein aggregate. Pchlide eluted in two peaks, one of which co-eluted with the enzyme activity peak. Corresponding column fractions from pc-1 y-7 showed similar protein and Pchlide elution profiles, but no enzyme activity. These results suggest that NADPH: Pchlide oxidoreductase is present, but inactive, in the double mutant, and are consistent with the hypothesis that the pc-1 locus codes for the enzyme.