Influence of the 9-methyl group of the retinal on the photocycle of bacteriorhodopsin studied by time-resolved rapid-scan and static low-temperature Fourier transform infrared difference spectroscopy.
The photocycle of bacteriorhodopsin (BR) regenerated with all-trans-9-demethylretinal was investigated by time-resolved rapid-scan Fourier transform infrared difference spectroscopy, by static low-temperature difference spectroscopy at 80, 170, and 213 K and by static steady-state difference spectroscopy at 278 K. In addition, the formation and decay of M intermediate was monitored at 412 nm with conventional flash photolysis experiments. Our data show that the removal of the 9-methyl group strongly changes the photocycle of BR. The reaction cycle is slowed down about 250-fold. The photoreaction is characterized by a slow rise of the M intermediate and by a very long-lived N intermediate. No O intermediate could be observed. The low-temperature spectra indicate that already at 80 K a KL-like photoproduct is formed. L can be obtained as in native BR at 170 K, but its decay appears to be inhibited, since it can still be observed at 213 K and high pH, in addition to the M intermediate. As in native BR, the 15-hydrogen out-of-plane modes of the L and N intermediates (observed in 2H2O) are very similar. Evidence for the existence of three N substates which differ in the protonation state of Asp96 and in the amide I bands is presented. This is explained by the extremely slowed-down reisomerization of the chromophore. The results are discussed with respect to alterations in the chromophore-protein interaction, caused by the removal of the 9-methyl group.
Weidlich
O
O
Institut für Biophysik und Strahlenbiologie, Albert-Ludwigs-Universität, Freiburg, Germany.
Friedman
N
N
Sheves
M
M
Siebert
F
F
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D008745:Methylation; D011487:Protein Conformation; D012172:Retinaldehyde / Q000031:analogs & derivatives* / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013816:Thermodynamics; D013997:Time Factors
C087891:9-desmethylretinal; D001436:Bacteriorhodopsins; D012172:Retinaldehyde