The enzyme protochlorophyllide oxidoreductase (POR, EC 1.3.1.33) has a key role in plant development. It catalyzes one of the later steps in chlorophyll synthesis, the light-induced reduction of protochlorophyllide (PChlide) into chlorophyllide (Chlide) in the presence of NADPH. Two isozymes of plant POR, POR A and POR B from barley, which differ in their function during plant life, are compared with respect to their substrate binding affinity, catalytic efficiency, and catalytic mechanism. POR B as compared to POR A shows an 5-fold higher binding affinity for protochlorophyllide a (PChlide) and an about 6-fold higher catalytic efficiency measured as ratio kcat/KM. Based on the reaction intermediates, which can be trapped at low temperatures the same reaction mechanism operates in both POR A and POR B. In contrast to results reported for POR enzymes from cyanobacteria, the initial light-driven step, which occurs at temperatures below 180 K already involves the full chemistry of the photoreduction and yields the reaction product, Chlide, in an enzyme-bound form. The subsequent dark reactions, which include cofactor (NADP(+)) release and cofactor (NADPH) rebinding, show different temperature dependencies for POR A and POR B and suggest a higher conformational flexibility of POR B in the surrounding of the active center. Both the higher substrate binding affinity and well-adapted enzyme dynamics are held responsible for the increased catalytic activity of POR B as compared to POR A.