Protochlorophyllide oxidoreductase: "dark" reactions of a light-driven enzyme

The light-driven enzyme NADPH:protochlorophyllide oxidoreductase (POR) catalyzes the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), a key regulatory step in the chlorophyll biosynthesis pathway. As POR is light activated, it allows intermediates in the reaction pathway to be observed by initiating catalysis with illumination at low temperatures, a technique that has recently been used to study the initial photochemistry. Here, we use low-temperature spectroscopy to show that the catalytic mechanism of POR involves two additional steps, which do not require light and have been termed the "dark" reactions. The first of these involves the conversion of the product of the initial light-driven reaction, a nonfluorescent radical species, into a new intermediate that has an absorbance maximum at 681 nm and a fluorescence peak at 684 nm. During the second dark step this species gradually blue shifts to yield the product, Chlide. The temperature dependence for each of these two processes was measured; the data revealed that these steps could only occur close to or above the "glass transition" temperature of proteins, suggesting that domain movements and/or reorganization of the protein are required for these stages of the catalytic mechanism.