Deuterium isotope effects in the photocycle transitions of the photoactive yellow protein.
The Photoactive Yellow Protein (PYP) from Halorhodospira halophila (formerly Ectothiorhodospira halophila) is increasingly used as a model system. As such, a thorough understanding of the photocycle of PYP is essential. In this study we have combined information from pOH- (or pH-) dependence and (kinetic) deuterium isotope effects to elaborate on existing photocycle models. For several characteristics of PYP we were able to make a distinction between pH- and pOH-dependence, a nontrivial distinction when comparing data from samples dissolved in H(2)O and D(2)O. It turns out that most characteristics of PYP are pOH-dependent. We confirmed the existence of a pB' intermediate in the pR to pB transition of the photocycle. In addition, we were able to show that the pR to pB' transition is reversible, which explains the previously observed biexponential character of the pR-to-pB photocycle step. Also, the absorption spectrum of pB' is slightly red-shifted with respect to pB. The recovery of the pG state is accompanied by an inverse kinetic deuterium isotope effect. Our interpretation of this is that before the chromophore can be isomerized, it is deprotonated by a hydroxide ion from solution. From this we propose a new photocycle intermediate, pB(deprot), from which pG is recovered and which is in equilibrium with pB. This is supported in our data through the combination of the observed pOH and pH dependence, together with the kinetic deuterium isotope effect.
Hendriks
Johnny
J
Department of Biochemistry and Molecular Biology, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
van Stokkum
Ivo H M
IH
Hellingwerf
Klaas J
KJ
D001426:Bacterial Proteins / Q000737:chemistry* / Q000528:radiation effects*; D003198:Computer Simulation; D003903:Deuterium / Q000737:chemistry*; D004735:Energy Transfer; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D007554:Isotopes / Q000737:chemistry; D007700:Kinetics; D008027:Light; D008956:Models, Chemical; D010777:Photochemistry / Q000379:methods; D020283:Photoreceptors, Microbial / Q000737:chemistry* / Q000528:radiation effects*; D011487:Protein Conformation / Q000528:radiation effects; D012996:Solutions / Q000737:chemistry; D013057:Spectrum Analysis / Q000379:methods*; D014466:Ultraviolet Rays; D014867:Water / Q000737:chemistry
D001426:Bacterial Proteins; D007554:Isotopes; D020283:Photoreceptors, Microbial; D012996:Solutions; C050162:photoactive yellow protein, Bacteria; D014867:Water; D003903:Deuterium