Cryogenic and Laser Photoexcitation Studies Identify Multiple Roles for Active Site Residues in the Light-driven Enzyme Protochlorophyllide Oxidoreductase

Abstract

The light-activated enzyme NADPH-protochlorophyllide oxidoreductase (POR) catalyzes the trans addition of hydrogen across the C-17-C-18 double bond of protochlorophyllide (Pchlide), a key step in chlorophyll biosynthesis. Similar to other members of the short chain alcohol dehydrogenase/reductase family of enzymes, POR contains a conserved Tyr and Lys residue in the enzyme active site, which are implicated in a proposed reaction mechanism involving proton transfer from the Tyr hydoxyl group to Pchlide. We have analyzed a number of POR variant enzymes altered in these conserved residues using a combination of steady-state turnover, laser photoexcitation studies, and low temperature fluorescence spectroscopy. None of the mutations completely abolished catalytic activity. We demonstrate their importance to catalysis by defining multiple roles in the overall reaction pathway. Mutation of either residue impairs formation of the ground state ternary enzyme-substrate complex, pointing to a key role in substrate binding. By analyzing the most active variant (Y193F), we show that Tyr-193 participates in proton transfer to Pchlide and stabilizes the Pchlide excited state, enabling hydride transfer from NADPH to Pchilde. Thus, in addition to confirming the probable identity of the proton donor in Pchlide reduction, our work defines additional roles for these residues in facilitating hydride transfer through stabilization of the ground and excited states of the ternary enzyme complex.

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

“…The NADPH-binding site is highly conserved in all PORs (Fig. 2A), and single residue changes in this region of the enzyme are known to compromise hydride transfer by substantially affecting the lifetime of the photoexcited state (15,33,34). This is consistent with the geometry and active site dynamics being optimally configured in all POR enzymes to enable efficient photochemistry.…”

“…The reaction chemistry is controlled by localized dynamics in all PORs through conser- vation of protein structure in the enzyme active site. The stringent structural requirements of the hydride transfer chemistry are consistent with the known sensitivity of this reaction to minor active site structural perturbations (15,33,34).…”

“…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). In POR from Thermosynechococcus elongatus, the hydride and proton transfer reactions occur sequentially by dynamically coupled nuclear tunneling (12).…”

Cell Growth Defect Factor1/CHAPERONE-LIKE PROTEIN OF POR1 Plays a Role in Stabilization of Light-Dependent Protochlorophyllide Oxidoreductase in Nicotiana benthamiana and Arabidopsis

“…The conserved Tyr and Lys residues in the active site of POR are involved in the formation of ternary enzyme-substrate complexes. Furthermore, the Tyr residue stabilizes the Pchlide excited state, promoting hydride transfer from NADPH to Pchlide, and participates in proton transfer (Menon et al, 2009). It has been generally thought that POR evolved from cyanobacteria, and the evolution of POR reflects the transformation from anoxygenic to oxygenic photosynthesis (Reinbothe et al, 1996(Reinbothe et al, , 2010Masuda and Takamiya, 2004).…”

“…POR catalyzes sequential hydride and proton transfers in the photoexcited and ground states, respectively (Menon et al, 2009;Heyes et al, 2011). The conserved Tyr and Lys residues in the active site of POR are involved in the formation of ternary enzyme-substrate complexes.…”