title: Protein-induced excited-state dynamics of protochlorophyllide.
abstract: The light-driven NADPH:protochlorophyllide oxidoreductase (POR) is a key enzyme of chlorophyll biosynthesis in angiosperms. POR's unique requirement for light to become catalytically active makes the enzyme an attractive model to study the dynamics of enzymatic reactions in real time. Here, we use picosecond time-resolved fluorescence and femtosecond pump-probe spectroscopy to examine the influence of the protein environment on the excited-state dynamics of the substrate, protochlorophyllide (PChlide), in the enzyme/substrate (PChlide/POR) and pseudoternary complex including the nucleotide cofactor NADP(+) (PChlide/NADP(+)/ POR). In comparison with the excited-state processes of unbound PChlide, the lifetime of the thermally equilibrated S(1) excited state is lengthened from 3.4 to 4.4 and 5.4 ns in the PChlide/POR and PChlide/NADP(+)/POR complex, whereas the nonradiative rates are decreased by ∼30 and 40%, respectively. This effect is most likely due to the reduced probability of nonradiative decay into the triplet excited state, thus keeping the risk of photosensitized side reactions in the enzyme low. Further, the initial reaction path involves the formation of an intramolecular charge-transfer state (S(ICT)) as an intermediate product. From a strong blue shift in the excited-state absorption, it is concluded that the S(ICT) state is stabilized by local interactions with specific protein sites in the catalytic pocket. The possible relevance of this result for the catalytic reaction in the enzyme POR is discussed.
authors.affiliation: Institute for Physical Chemistry and Abbe Centre of Photonics, Friedrich-Schiller-University Jena, Helmholtzweg 4, D-07743 Jena, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Hanf
authors.forename: Sonja
authors.initials: S
authors.lastname: Fey
authors.forename: Benjamin
authors.initials: B
authors.lastname: Dietzek
authors.forename: Michael
authors.initials: M
authors.lastname: Schmitt
authors.forename: Christiane
authors.initials: C
authors.lastname: Reinbothe
authors.forename: Steffen
authors.initials: S
authors.lastname: Reinbothe
authors.forename: Gudrun
authors.initials: G
authors.lastname: Hermann
authors.forename: Jürgen
authors.initials: J
authors.lastname: Popp
mesh_terms: D055162:Biocatalysis / Q000187:drug effects / Q000528:radiation effects; D007700:Kinetics; D008027:Light; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry; D010777:Photochemistry; D010860:Pigments, Biological / Q000737:chemistry; D011506:Proteins / Q000737:chemistry* / Q000494:pharmacology; D011521:Protochlorophyllide / Q000737:chemistry* / Q000528:radiation effects
chemical_list: D010860:Pigments, Biological; D011506:Proteins; D011521:Protochlorophyllide; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase