Abstract
Experimental interrogation of the relationship between protein dynamics and enzyme catalysis is challenging. Light-activated protochlorophyllide oxidoreductase (POR) is an excellent model for investigating this relationship because photoinitiation of the reaction cycle enables coordinated turnover in a "dark-assembled" ternary enzyme-substrate complex. The catalytic cycle involves sequential hydride and proton transfers (from NADPH and an active site tyrosine residue, respectively) to the substrate protochlorophyllide. Studies with a limited cross-species subset of POR enzymes (n = 4) have suggested that protein dynamics associated with hydride and proton transfer are distinct [Heyes, D. J., Levy, C., Sakuma, M., Robertson, D. L., and Scrutton, N. S. (2011) J. Biol. Chem. 286, 11849-11854]. Here, we use steady-state assays and single-turnover laser flash spectroscopy to analyze hydride and proton transfer dynamics in an extended series of POR enzymes taken from many species, including cyanobacteria, algae, embryophytes, and angiosperms. Hydride/proton transfer in all eukaryotic PORs is faster compared to prokaryotic PORs, suggesting active site architecture has been optimized in eukaryotic PORs following endosymbiosis. Visible pump-probe spectroscopy was also used to demonstrate a common photoexcitation mechanism for representative POR enzymes from different branches of the phylogenetic tree. Dynamics associated with hydride transfer are localized to the active site of all POR enzymes and are conserved. However, dynamics associated with proton transfer are variable. Protein dynamics associated with proton transfer are also coupled to solvent dynamics in cyanobacterial PORs, and these networks are likely required to optimize (shorten) the donor-acceptor distance for proton transfer. These extended networks are absent in algal and plant PORs. Our analysis suggests that extended networks of dynamics are disfavored, possibly through natural selection. Implications for the evolution of POR and more generally for other enzyme catalysts are discussed.
Highlights
A phylogenetic analysis of the available gene sequences for protochlorophyllide oxidoreductase (POR) shows broad separation into these two groups (Figure 2), the cyanobacterial group is more diverse than the plant group, indicated by the longer branch lengths
The catalytic activities of all POR enzymes were measured under steady-state conditions to determine the kinetic parameters for the reaction (Table 1)
We have conducted a more comprehensive cross-species analysis of the dynamics required for catalysis in the light-activated POR enzyme
Summary
E nzymes are dynamic molecules, and understanding the role of protein dynamics associated with bond breaking and/or making is important.[2−9] Answers to a number of key questions are needed, including the following: (i) Do conformational fluctuations within an enzyme active site couple to longer-range structural variations?10−13 (ii) Are the dynamic profiles of enzyme homologues conserved alongside reaction chemistry, or is there variability?4 (iii) Are the dynamic profiles of enzyme homologues influenced by natural selection? The solvent environment is coupled to the enzyme, and any influence of solvent dynamics on reaction rate can be probed by varying solution viscosity.[7,14−17] Solution visocity effects, on either enzyme turnover (kcat) or the rate constants for individual steps of the catalytic cycle, should inform on the conservation (or otherwise) of dynamics in enzymes that catalyze the same chemistry but are from different species. Article subsequent transfer of a proton from a conserved tyrosine residue to form Chlide product (Figure 1).[24,25] Both the hydride and proton transfer reactions proceed by quantum mechanical tunneling.[23,26] A role for fast (so-called promoting) motions has been inferred for both hydride and proton transfer reactions catalyzed by POR.[7,23] A series of ordered product release and coenzyme binding steps have been shown to follow hydride and proton transfer, and these are required to complete the catalytic cycle. We expand on this analysis by investigating the dynamics of hydride and proton transfer in PORs from a wide range of cyanobacteria, algae, liverwort, moss, and higher plants
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