Resonance Raman study of halorhodopsin photocycle kinetics, chromophore structure, and chloride-pumping mechanism.
Kinetic resonance Raman spectra of the HR520, HR640, and HR578 species in the halorhodopsin photocycle are obtained using time delays ranging from 5 microseconds to 10 ms in 0.3 M NO3-, 0.3 M Cl-, and 3 M Cl-. The Raman intensities are converted to absolute concentrations by using a conservation of molecules constraint. The simplest kinetic scheme that satisfactorily models the data is HR578-->HR520 in equilibrium with HR640-->HR578. The rate constant for the HR640-->HR578 transition increases with Cl- concentration, suggesting that Cl- is taken up between HR640 and HR578. The ratio of the forward to the reverse rate constants connecting HR520 and HR640 increases as the inverse of the Cl- concentration, suggesting that Cl- is released during the HR520-->HR640 step. The configuration about the C13 = C14 bond of the retinal chromophore in HR640 is examined by regenerating the protein with [12,14-2H2]retinal. The C12-2H + C14-2H rocking vibration for HR640 is observed at 943 cm-1, demonstrating that the chromophore is 13-cis. The changes in the resonance Raman spectrum of HR640 in response to 2H2O suspension indicates that the Schiff base linkage to the protein is protonated. None of the HR640 fingerprint vibrations shift significantly in 2H2O, suggesting that the Schiff base adopts a C = N anti configuration; this assignment is supported by the frequency of the C15-2H rocking mode (1002 cm-1). The 13-cis structure for the chromophore in HR640 requires that thermal isomerization back to all-trans occurs in the HR640-->HR578 transition. These structural and kinetic results are incorporated into a two-state C-T model for Cl- pumping.
Ames
J B
JB
Department of Chemistry, University of California, Berkeley 94720.
Raap
J
J
Lugtenburg
J
J
Mathies
R A
RA
D001436:Bacteriorhodopsins / Q000737:chemistry*; D002712:Chlorides / Q000378:metabolism*; D019617:Halobacterium salinarum / Q000737:chemistry / Q000378:metabolism; D025602:Halorhodopsins; D007700:Kinetics; D008956:Models, Chemical; D010777:Photochemistry; D013059:Spectrum Analysis, Raman
D002712:Chlorides; D025602:Halorhodopsins; D001436:Bacteriorhodopsins