The enzyme protochlorophyllide oxidoreductase (POR) catalyses a lightdependent step in chlorophyll biosynthesis that is essential to photosynthesis and ultimately all life on Earth. 1-3 POR, which is one of three known light-dependent enzymes, 4,5 catalyzes reduction of the photosensitizer and substrate protochlorophyllide to form the pigment chlorophyllide. Despite its biological importance, a structural basis for POR photocatalysis has remained elusive. Here, we report crystal structures of cyanobacterial PORs from Thermosynechococcus elongatus and Synechocystis sp. in their free forms, and in complex with nicotinamide coenzyme. Our structural models and simulations of the ternary protochlorophyllide-NADPH-POR complex have identified multiple interactions in the POR active site that are important for protochlorophyllide binding, photosensitization and photochemical conversion to chlorophyllide. We demonstrate the importance of active-site architecture and protochlorophyllide structure in experiments using POR variants and protochlorophyllide analogues. These studies reveal how the POR active site facilitates light-driven reduction of protochlorophyllide by localized hydride transfer from NADPH and long-range proton transfer along structurally defined proton-transfer pathways. As the light-driven step in the chlorophyll biosynthetic pathway (Fig. 1), the POR reaction acts as the trigger for the germination of seedlings =in plants and provokes a marked change in the morphological development of the plant. 2,3 Given this crucial biological role, POR has been the focus of numerous mechanistic and biophysical investigations. A combination of time-resolved (at the femtosecond-to-second scale) and cryogenic spectroscopy methods have provided some understanding of the mechanism of POR photocatalysis in a range of photosynthetic organisms, including cyanobacteria and plants. Picosecond excited-state dynamics in the protochlorophyllide (Pchlide) molecule are thought to result in excited state interactions between the substrate and active-site residues that are necessary to trigger the subsequent reaction chemistry. 6-12 This involves sequential transfer of a hydride equivalent from NADPH and a proton transfer from either an active site residue or solvent. Proton transfer is reliant on solvent dynamics and an implied network of extended protein motions that occur on the microsecond timescale. 13-17 Hydride transfer from NADPH is not concerted, but occurs in a stepwise manner that involves
Strategy for bio-alkane gas (propane and butane) production through the conversion of waste volatile fatty acids by bacterial cultures.
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