TIME-RESOLVED TITRATIONS OF THE SCHIFF-BASE AND OF THE ASP(85) RESIDUE IN ARTIFICIAL BACTERIORHODOPSINS

被引:19
作者
DRUCKMANN, S
OTTOLENGHI, M
ROUSSO, I
FRIEDMAN, N
SHEVES, M
机构
[1] HEBREW UNIV JERUSALEM,DEPT PHYS CHEM,IL-91904 JERUSALEM,ISRAEL
[2] WEIZMANN INST SCI,DEPT ORGAN CHEM,IL-76100 REHOVOT,ISRAEL
关键词
D O I
10.1021/bi00037a050
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
Deprotonation/protonation processes involving the retinal Schiff base and the Asp(85) residue play dominant roles in the light-induced proton pump of bacteriorhodopsin (bR). Although the pK(a) values of these two moieties in unphotolyzed bR are well established, the kinetics of the respective titrations in the native pigment are difficult to interpret, primarily due to the extreme (nonphysiological) pK(a) values of the two moieties (12.2 +/- 0.2 and 2.7, in 0.1 M NaCl, for the Schiff base and for Asp(85), respectively). These difficulties are circumvented by applying stopped-flow techniques, time resolving the titrations of several artificial bRs in which the pK(a) values of the above two residues are substantially modified: 13-CF3 bR, pK(a) (Schiff base) = 8.2 +/- 0.2; 13-demethyl-11,14-epoxy bR, pK(a) (Schiff base) = 8.2 +/- 0.1 (in 0.1 M NaCl); aromatic bR, pK(a) (Asp(85)) = 5.2 +/- 0.1 (in water). The R82Q bR mutant, pK(a) (Asp(85)) similar or equal to 7.2 was also employed. A major objective was to verify whether the basic relationships of homogeneous kinetics obeyed by elementary acid/base systems in solution (primarily, the possibility to express the equilibrium constant as the ratio of the forward and back rate constants) are also obeyed by the Schiff base and Asp(85) moieties. We found that this is the case for the Schiff base in the pH range between 7 and 9 but not at lower pH. These observations led to the conclusion that the Schiff base is titrable from the outside medium via a proton channel, which becomes saturated, and thus rate determining, below pH congruent to 7. The observed protonation rate constant in the pH = 7-9 range is k(a) = 6.0 x 10(7) M(-1) s(-1), implying a reactivity that is lower by 3 orders of magnitude as compared to the diffusion-controlled rate constant of an elementary acid/base in homogeneous solutions. In the case of Asp(85), ka could not be directly determined. The titration rates observed in the case of pigment IV are, however, consistent with a model in which the Schiff base and Asp(85) are exposed to the extracellular side via the same proton channel. It is suggested that the rate-determining step in proton translocation via this channel is a transfer between Asp(85) and the outside, rather than between Asp(85) and the Schiff base. This conclusion applies independently of whether Asp(85) is protonated or non-protonated. The results are relevant to basic questions related to the proton pump mechanism in bR, primarily (a) the exposure direction (to the outside or to the inside of the cell) of the Schiff base and of Asp(85) in, unphotolyzed bR and (b) the nature of the still unidentified protein residue (XH) whose proton is translocated to the outside during the bacteriorhodopsin photocycle. We conclude that, in variance with the Schiff base in unphotolyzed bR or with Asp(85) (in, photolyzed or unphotolyzed bR), during the photocycle the XH moiety is highly exposed to the outside medium. More generally, our study bears on the basic problem concerning the relationship between the kinetics of the titration of protein residues and their respective (''thermodynamic'') equilibrium constants.
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收藏
页码:12066 / 12074
页数:9
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