RESONANCE RAMAN-STUDY OF HALORHODOPSIN PHOTOCYCLE KINETICS, CHROMOPHORE STRUCTURE, AND CHLORIDE-PUMPING MECHANISM

被引:47
作者
AMES, JB
RAAP, J
LUGTENBURG, J
MATHIES, RA
机构
[1] UNIV CALIF BERKELEY,DEPT CHEM,BERKELEY,CA 94720
[2] LEIDEN UNIV,DEPT CHEM,2312 AV LEIDEN,NETHERLANDS
关键词
D O I
10.1021/bi00165a002
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Kinetic resonance Raman spectra of the HR520, HR640, and HR578 species in the halorhodopsin photocycle are obtained using time delays ranging from 5 mus 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 <-> 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 C-13=C-14 bond of the retinal chromophore in HR640 is examined by regenerating the protein with [12,14-H-2(2)]retinal. The C-12-H-2 + C-14-H-2 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 (H2O)-H-2 suspension indicates that the Schiff base linkage to the protein is protonated. None of the HR640 fingerprint vibrations shift significantly in (H2O)-H-2, suggesting that the Schiff base adopts a C=N anti configuration; this assignment is supported by the frequency of the C-15-H-2 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.
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收藏
页码:12546 / 12554
页数:9
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