Abstract

The chlorophyll biosynthesis enzyme protochlorophyllide reductase (POR) catalyzes the reduction of protochlorophyllide into chlorophyllide in the presence of NADPH. This reaction is light dependent and provides a unique opportunity to initiate enzyme catalysis with a short laser pulse and study subsequent reaction dynamics. We used pump-probe absorption spectroscopy to obtain time-resolved data on enzymatic proton and hydride transfer. A NADPH-PChlide-POR complex, PChlide in solution and a Y189F mutant lacking the putative proton donor Tyr189 were examined. Only in wild-type in the presence of NADPH, POR catalyzed the formation of the Chlide-product. We conclude that the light-activated reaction path is multi-dimensional and involves both a single-step proton/hydride mechanism occurring in ~3 ps and a sequential mechanism in which hydride transfer (~400ps) is rate limiting.

© 2002 Optical Society of America