Spectroscopic characterization of the photocycle intermediates of photoactive yellow protein.
The absorption spectra of photocycle intermediates of photoactive yellow protein mutants were compared with those of the corresponding intermediates of wild type to probe which amino acid residues interact with the chromophore in the intermediate states. B and H intermediates were produced by irradiation and trapped at 80 K, and L intermediates at 193 K. The absorption spectra of these intermediates produced from R52Q were identical to those from wild type, whereas those from E46Q and T50V were 7-15 nm red-shifted as those in the dark states. The absorption spectra of M intermediates were measured by flash photolysis at room temperature. Those of Y42F, T50V, and R52Q were identical to that of wild type, whereas that of E46Q was 11 nm red-shifted. Assuming that the intermediates of mutants have a structure comparable to that of wild type, these findings suggest the following: Glu46 interacts with the chromophore throughout the photocycle, interaction between the chromophore and Thr50 as well as Tyr42 is lost upon the formation of M intermediate, and Arg52 never interacts with the chromophore directly. The hydrogen-bonding network around the phenolic oxygen of the chromophore would be thus maintained until L intermediate decays, and the global conformational change would take place by the loss of the hydrogen bond between the chromophore and Tyr42. This model conflicts with some of the results of previous crystallographic studies, suggesting that the reaction mechanism in the crystal may be different from that in solution.
Imamoto
Y
Y
Graduate School of Materials Science, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan. imamoto@ms.aist-nara.ac.jp
Mihara
K
K
Tokunaga
F
F
Kataoka
M
M
D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D003080:Cold Temperature; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry; D010782:Photolysis; D020283:Photoreceptors, Microbial; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; C050162:photoactive yellow protein, Bacteria