Time-resolved FTIR spectroscopy of the photointermediates involved in fast transient H+ release by proteorhodopsin.
Proteorhodopsin (pR) is a homologue of bacteriorhodopsin (bR) that has been recently discovered in oceanic bacterioplankton. Like bR, pR functions as a light-driven proton pump. As previously characterized by laser flash induced absorption spectroscopy (Krebs, R. A.; Alexiev, U.; Partha, R.; DeVita, A. M.; Braiman, M. S. BMC Physiol. 2002, 2, 5), the pR photocycle shows evidence of light-induced H(+) release on the 10-50 micros time scale, and of substantial accumulation of the M intermediate, only at pH values above 9 and after reconstitution into phospholipid followed by extensive washing to remove detergent. We have therefore measured the time-resolved FTIR difference spectra of pR intermediates reconstituted into DMPC vesicles at pH 9.5. A mixture of K- and L-like intermediates, characterized by a 1516 cm(-1) positive band and a 1742 cm(-1) negative band respectively, appears within 20 micros after photolysis. This mixture decays to an M-like state, with a clear band at 1756 cm(-1) due to protonation of Asp-97. The 50-70 micros rise of M at pH 9.5 is similar to (but a little slower than) the rise times for M formation and H(+) release that were reported earlier based on flash photolysis measurements of pR reconstituted into phospholipids with shorter acyl chains. We conclude that, at pH 9.5, H(+) release occurs while Asp-97 is still protonated; i.e., this aspartic acid cannot be the H(+) release group observed by flash photolysis under similar conditions.
Xiao
Yaowu
Y
Department of Chemistry, Syracuse University, Syracuse, New York 13244-4100, USA.
Partha
Ranga
R
Krebs
Richard
R
Braiman
Mark
M
D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D010777:Photochemistry; D012243:Rhodopsin / Q000737:chemistry*; D025604:Rhodopsins, Microbial; D012680:Sensitivity and Specificity; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013997:Time Factors
D025604:Rhodopsins, Microbial; C413548:proteorhodopsin; D012243:Rhodopsin; D004134:Dimyristoylphosphatidylcholine