Stepwise Hydride Transfer in a Biological System: Insights into the Reaction Mechanism of the Light-Dependent Protochlorophyllide Oxidoreductase

Hydride transfer plays a crucial role in a wide range of biological systems. However, its mode of action (concerted or stepwise) is still under debate. Light-dependent NADPH: protochlorophyllide oxidoreductase (POR) catalyzes the stereospecific trans addition of a hydride anion and a proton across the C-17-C-18 double bond of protochlorophyllide. Time-resolved absorption and emission spectroscopy were used to investigate the hydride transfer mechanism in POR. Apart from excited states of protochlorophyllide, three discrete intermediates were resolved, consistent with a stepwise mechanism that involves an initial electron transfer from NADPH. A subsequent proton-coupled electron transfer followed by a proton transfer yield distinct different intermediates for wild type and the C226S variant, that is, initial hydride attaches to either C-17 or C-18, but ends in the same chlorophyllide stereoisomer. This work provides the first evidence of a stepwise hydride transfer in a biological system.