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

Abstract

Protein dynamics are crucial for realizing the catalytic power of enzymes, but how enzymes have evolved to achieve catalysis is unknown. The light-activated enzyme protochlorophyllide oxidoreductase (POR) catalyzes sequential hydride and proton transfers in the photoexcited and ground states, respectively, and is an excellent system for relating the effects of motions to catalysis. Here, we have used the temperature dependence of isotope effects and solvent viscosity measurements to analyze the dynamics coupled to the hydride and proton transfer steps in three cyanobacterial PORs and a related plant enzyme. We have related the dynamic profiles of each enzyme to their evolutionary origin. Motions coupled to light-driven hydride transfer are conserved across all POR enzymes, but those linked to thermally activated proton transfer are variable. Cyanobacterial PORs require complex and solvent-coupled dynamic networks to optimize the proton donor-acceptor distance, but evolutionary pressures appear to have minimized such networks in plant PORs. POR from Gloeobacter violaceus has features of both the cyanobacterial and plant enzymes, suggesting that the dynamic properties have been optimized during the evolution of POR. We infer that the differing trajectories in optimizing a catalytic structure are related to the stringency of the chemistry catalyzed and define a functional adaptation in which active site chemistry is protected from the dynamic effects of distal mutations that might otherwise impact negatively on enzyme catalysis.Currently, one of the most challenging questions in biology is how enzymes have evolved to optimize the dynamic processes that enable their extraordinary rate enhancements (1-8). The role of protein motions and mechanisms of coupling to active site chemistry and solvent dynamics have been debated extensively (1, 5-10), but how the intrinsic motions of enzyme molecules have been affected by millions of years of evolutionary pressure (and the influence these motions have on catalysis) remains an open question. An evolutionary perspective of protein dynamics can be acquired only by considering functional differences between enzymes from species that span the evolutionary time scale. Hence, we have now studied this problem in the light-activated enzyme protochlorophyllide oxidoreductase (POR 3 ; EC 1.3.1.33), which is an excellent system for relating the effects of motions to catalysis in the context of proton and hydride transfer chemistry (11,12).POR catalyzes the light-dependent trans-addition of hydrogen across the C17-C18 double bond of the D-ring of protochlorophyllide (Pchlide) to produce chlorophyllide, an essential step in the synthesis of chlorophyll, the most abundant pigment on Earth (Fig. 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...

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

“…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). When the catalytic mechanism was compared between cyanobacterial and plant POR, the light-driven hydride transfer step was found to be conserved among all POR enzymes, but the proton transfer step is variable, suggesting that functional adaptation and optimization occurred during the evolution of POR (Heyes et al, 2011). CPP1 homologs are ubiquitously found in oxygenic photosynthetic organisms that possess POR, including cyanobacteria, green algae, mosses, gymnosperms, and angiosperms.…”

“…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.…”

Identification of a Long-range Protein Network That Modulates Active Site Dynamics in Extremophilic Alcohol Dehydrogenases

“…Generally, a plot of ln(k) against 1/T yields a straight line. However, for some enzymes, a transition to more sluggish catalysis has been seen to occur abruptly below a threshold temperature in a manner that is distinct from either a change in the rate-determining step or a decrease in enzyme stability (5,24,26). All of these enzymes and most other enzymes that have been shown to exhibit Arrhenius breakpoints are multimeric (see for example, Refs.…”

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

“…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.…”

“…[9] Conventional ultrafast pump-visible absorption-probe measurements over timescales up to % 3nsw ere coupled to those using at ransient absorption spectrometer that uses an electronically controlled pump-probe delay to provide continuous temporal coverage from picosecond to microsecond. Analogous measurements in the IR region were carried out using the recently developed time-resolved multiple probe spectroscopy (TR M PS) technique,w here electrical timing again provides nanosecond-to-microsecond delay control of the first probe pulse,c ombined with the optical delay limits of afew nanoseconds.…”