The First Catalytic Step of the Light-driven Enzyme Protochlorophyllide Oxidoreductase Proceeds via a Charge Transfer Complex

Abstract

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 (A 696 ). 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 A 696 . 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 A 696 formation and NADPH oxidation are identical, and we propose that formation of the A 696 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 A 696 charge transfer intermediate facilitates transfer of a proton to the C-18 of Pchlide during the subsequent first "dark" reaction.

Excited‐State Charge Separation in the Photochemical Mechanism of the Light‐Driven Enzyme Protochlorophyllide Oxidoreductase

“…In the wild-type POR ternary enzyme-substrate complex the formation of the hydride transfer intermediate,w ith ac haracteristic absorbance band at 696 nm, [4][5][6] is observed with alifetime of approximately 500 ns ( Figure 3A), which is similar to that reported previously. [6] In contrast, the same intermediate is not found in the Y193F sample,c onfirming that photochemistry is impaired in this variant.…”

“…Consequently,P OR has become an important model system for studying many aspects of enzyme catalysis. [2][3][4][5][6][7][8][9] Ther eaction catalyzed by POR involves ah ighly endergonic light-driven hydride transfer from the pro-S face of the nicotinamide ring of NADPH to the C17 position of the Pchlide molecule, [5,6] followed by an exergonic thermally activated proton transfer, most likely from ac onserved Tyr residue,t ot he C18 position of Pchlide [9] (Figure 1). The hydride and proton transfer reactions occur in as equential mechanism on the microsecond timescale by nuclear tunneling.…”

Nuclear Quantum Tunneling in the Light-activated Enzyme Protochlorophyllide Oxidoreductase

“…However, despite its biological significance, the detailed mechanism of this light-activated catalysis remains poorly understood. Low temperature spectroscopy has identified a number of steps in the reaction cycle, including an initial light-driven reaction (11,12) and subsequent dark reactions (13)(14)(15). In the reaction, reduction of Pchlide by NADPH in the POR-NADPH-Pchlide complex involves hydride transfer from the pro-S face of NADPH to the C-17 atom of Pchlide (16).…”

A Twin-track Approach Has Optimized Proton and Hydride Transfer by Dynamically Coupled Tunneling during the Evolution of Protochlorophyllide Oxidoreductase

“…1) (11,13). The reaction involves a highly endergonic (ground state to excited state) light-driven hydride transfer from the pro-S face of the nicotinamide ring of NADPH to C17 of the Pchlide molecule (12,14), followed by an exergonic (ground state) proton transfer from a conserved Tyr residue to C18 ( Fig. 1) (15).…”