Abstract
In chlorophyll biosynthesis, the light-activated enzyme protochlorophyllide oxidoreductase catalyzes trans addition of hydrogen across the C-17-C-18 double bond of the chlorophyll precursor protochlorophyllide (Pchlide). This unique light-driven reaction plays a key role in the assembly of the photosynthetic apparatus, but despite its biological importance, the mechanism of light-activated catalysis is unknown. In this study, we show that Pchlide reduction occurs by dynamically coupled nuclear quantum tunneling of a hydride anion followed by a proton on the microsecond time scale in the Pchlide excited and ground states, respectively. We demonstrate the need for fast dynamic searches to form degenerate "tunneling-ready" configurations within the lifetime of the Pchlide excited state from which hydride transfer occurs. Moreover, we have found a breakpoint at -27 degrees C in the temperature dependence of the hydride transfer rate, which suggests that motions/vibrations that are important for promoting light-activated hydride tunneling are quenched below -27 degrees C. We observed no such breakpoint for the proton-tunneling reaction, indicating a reliance on different promoting modes for this reaction in the enzyme-substrate complex. Our studies indicate that the overall photoreduction of Pchlide is endothermic and that rapid dynamic searches are required to form distinct tunneling-ready configurations within the lifetime of the photoexcited state. Consequently, we have established the first important link between photochemical and nuclear quantum tunneling reactions, linked to protein dynamics, in a biologically significant system.
Highlights
Domain) facilitate the H-tunneling reactions remains elusive (4 – 6)
POR catalyzes the trans addition of hydrogen across the C-17–C-18 double bond of the chlorophyll precursor protochlorophyllide (Pchlide) to produce chlorophyllide (Chlide) [7], a unique light-driven reaction in the synthesis of the most abundant pigment on earth, which plays a key role in the assembly of the photosynthetic apparatus [8, 9]
The theoretical model for the reduction of the driven chemical steps is currently lacking. To access this chem- C-17–C-18 bond of Pchlide contains the pigment without side istry we have synchronized the turnover of the POR- chains, except for the propionate chain that is attached to C-17, NADPH-Pchlide complex by using a which is replaced by an ethyl group to keep the overall charge of
Summary
Domain) facilitate the H-tunneling reactions remains elusive (4 – 6). A major limitation has been the inability to synchronously trigger catalysis on ultrafast time scales for the majority of enzymes that require mixing strategies to initiate the reaction. We have combined kinetic analysis with studies the model, and they function as hydride and proton donors, of both the temperature and isotopic dependence of the rate of respectively.
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