The first catalytic step of the light-driven enzyme protochlorophyllide oxidoreductase proceeds via a charge transfer complex

In chlorophyll biosynthesis protochlorophyllide reductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide) to chlorophyllide, providing a rare opportunity to trap and characterize catalytic intermediates at low temperatures. Moreover, the presence of a chlorophyll-like molecule allows the use of EPR, electron nuclear double resonance, and Stark spectroscopies, previously used for the analysis of photosynthetic systems, to follow catalytic events in the active site of POR. Different models involving the formation of either radical species or charge transfer complexes have been proposed for the initial photochemical step, which forms a nonfluorescent intermediate absorbing at 696 nm (A696). Our EPR data show that the concentration of the radical species formed in the initial photochemical step is not stoichiometric with conversion of substrate. Instead, a large Stark effect, indicative of charge transfer character, is associated with A696. Two components were required to fit the Stark data, providing clear evidence that charge transfer complexes are formed during the initial photochemistry. The temperature dependences of both A696 formation and NADPH oxidation are identical, and we propose that formation of the A696 state involves hydride transfer from NADPH to form a charge transfer complex. A catalytic mechanism of POR is suggested in which Pchlide absorbs a photon, creating a transient charge separation across the C-17-C-18 double bond, which promotes ultrafast hydride transfer from the pro-S face of NADPH to the C-17 of Pchlide. The resulting A696 charge transfer intermediate facilitates transfer of a proton to the C-18 of Pchlide during the subsequent first "dark" reaction.