The photochemical reaction cycle and photoinduced proton transfer of sensory rhodopsin II (Phoborhodopsin) from Halobacterium salinarum.
Sensory rhodopsin II (HsSRII, also called phoborhodopsin) is a negative phototaxis receptor of Halobacterium salinarum, a bacterium that avoids blue-green light. In this study, we expressed the protein in Escherichia coli cells, and reconstituted the purified protein with phosphatidylcholine. The reconstituted HsSRII was stable. We examined the photocycle by flash-photolysis spectroscopy in the time range of milliseconds to seconds, and measured proton uptake/release using a transparent indium-tin oxide electrode. The pKa of the counterion of the Schiff base, Asp(73), was 3.0. Below pH 3, the depleted band was observed on flash illumination, but the positive band in the difference spectra was not found. Above pH 3, the basic photocycle was HsSRII (490) --> M (350) --> O (520) --> Y (490) --> HsSRII, where the numbers in parentheses are the maximum wavelengths. The decay rate of O-intermediate and Y-intermediate were pH-independent, whereas the M-intermediate decay was pH-dependent. For 3 < pH < 4.5, the M-decay was one phase, and the rate decreased with an increase in pH. For 4.5 < pH < 6.5, the decay was one phase with pH-independent rates, and azide markedly accelerated the M-decay. These findings suggest the existence of a protonated amino acid residue (X-H) that may serve as a proton relay to reprotonate the Schiff base. Above pH 6.5, the M-decay showed two phases. The fast M-decay was pH-independent and originated from the molecule having a protonated X-H, and the slow M-decay originated from the molecule having a deprotonated X, in which the proton came directly from the outside. The analysis yielded a value of 7.5 for the pKa of X-H. The proton uptake and release occurred during M-decay and O-decay, respectively.
Tamogami
Jun
J
Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan; Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Kikukawa
Takashi
T
Ikeda
Yoichi
Y
Takemura
Ayaka
A
Demura
Makoto
M
Kamo
Naoki
N
D001224:Aspartic Acid / Q000737:chemistry; D001436:Bacteriorhodopsins / Q000378:metabolism; D004566:Electrodes; D004926:Escherichia coli / Q000201:enzymology; D019617:Halobacterium salinarum / Q000378:metabolism*; D025602:Halorhodopsins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007204:Indium / Q000737:chemistry; D007477:Ions; D008027:Light; D010777:Photochemistry / Q000379:methods*; D011522:Protons; D025603:Sensory Rhodopsins / Q000737:chemistry*; D013053:Spectrophotometry / Q000379:methods; D017971:Tin Compounds / Q000737:chemistry
D025602:Halorhodopsins; D007477:Ions; D011522:Protons; D025603:Sensory Rhodopsins; D017971:Tin Compounds; C061508:sensory rhodopsin II protein, archaeal; D007204:Indium; D001224:Aspartic Acid; D001436:Bacteriorhodopsins; C045358:stannic oxide