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 C₁₇?C₁₈ 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 C₁₇ or C₁₈, but ends in the same chlorophyllide stereoisomer. This work provides the first evidence of a stepwise hydride transfer in a biological system.