title: Simultaneous measurements of solvent dynamics and functional kinetics in a light-activated enzyme.
abstract: Solvent fluctuations play a key role in controlling protein motions and biological function. Here, we have studied how individual steps of the reaction catalyzed by the light-activated enzyme protochlorophyllide oxidoreductase (POR) couple with solvent dynamics. To simultaneously monitor the catalytic cycle of the enzyme and the dynamical behavior of the solvent, we designed temperature-dependent UV-visible microspectrophotometry experiments, using flash-cooled nanodroplets of POR to which an exogenous soluble fluorophore was added. The formation and decay of the first two intermediates in the POR-catalyzed reaction were measured, together with the solvent glass transition and the buildup of crystalline ice at cryogenic temperatures. We find that formation of the first intermediate occurs below the glass transition temperature (T(g)), and is not affected by changes in solvent dynamics induced by modifying the glycerol content. In contrast, formation of the second intermediate occurs above T(g) and is influenced by changes in glycerol concentration in a manner remarkably similar to the buildup of crystalline ice. These results suggest that internal, nonslaved protein motions drive the first step of the POR-catalyzed reaction whereas solvent-slaved motions control the second step. We propose that the concept of solvent slaving applies to complex enzymes such as POR.
authors.affiliation: Institut de Biologie Structurale Jean-Pierre Ebel, Centre d'Etudes Atomiques, Centre National de la Recherche Scientifique, Université Joseph Fourier, Grenoble, France.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Durin
authors.forename: Aude
authors.initials: A
authors.lastname: Delaunay
authors.forename: Claudine
authors.initials: C
authors.lastname: Darnault
authors.forename: Derren J
authors.initials: DJ
authors.lastname: Heyes
authors.forename: Antoine
authors.initials: A
authors.lastname: Royant
authors.forename: Xavier
authors.initials: X
authors.lastname: Vernede
authors.forename: C Neil
authors.initials: CN
authors.lastname: Hunter
authors.forename: Martin
authors.initials: M
authors.lastname: Weik
authors.forename: Dominique
authors.initials: D
authors.lastname: Bourgeois
mesh_terms: D000465:Algorithms; D001426:Bacterial Proteins / Q000737:chemistry; D000458:Cyanobacteria / Q000201:enzymology; D005454:Fluorescence Polarization; D007700:Kinetics; D008027:Light; D015591:Microspectrophotometry / Q000379:methods; D008956:Models, Chemical; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry*; D011487:Protein Conformation; D012997:Solvents / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013696:Temperature; D014783:Viscosity
chemical_list: D001426:Bacterial Proteins; D012997:Solvents; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase