Hydride transfers play 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. We used timeresolved absorption and emission spectroscopies to investigate the hydride transfer mechanism in POR. Apart from excited states of protochlorophyllide, we resolved three discrete intermediates consistent with a stepwise mechanism that involves an initial electron transfer from NADPH. Following proton coupled electron transfer and
a subsequent proton transfer yield distinct different intermediates for wild type and the C226S variant, i.e. 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.