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

Citation
S. Druckmann et al., TIME-RESOLVED TITRATIONS OF THE SCHIFF-BASE AND OF THE ASP(85) RESIDUE IN ARTIFICIAL BACTERIORHODOPSINS, Biochemistry, 34(37), 1995, pp. 12066-12074
Citations number
39
Categorie Soggetti
Biology
Journal title
ISSN journal
00062960
Volume
34
Issue
37
Year of publication
1995
Pages
12066 - 12074
Database
ISI
SICI code
0006-2960(1995)34:37<12066:TTOTSA>2.0.ZU;2-3
Abstract
Deprotonation/protonation processes involving the retinal Schiff base and the Asp(85) residue play dominant roles in the light-induced proto n pump of bacteriorhodopsin (bR). Although the pK(a) values of these t wo moieties in unphotolyzed bR are well established, the kinetics of t he respective titrations in the native pigment are difficult to interp ret, primarily due to the extreme (nonphysiological) pK(a) values of t he two moieties (12.2 +/- 0.2 and 2.7, in 0.1 M NaCl, for the Schiff b ase and for Asp(85), respectively). These difficulties are circumvente d by applying stopped-flow techniques, time resolving the titrations o f several artificial bRs in which the pK(a) values of the above two re sidues 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 wat er). The R82Q bR mutant, pK(a) (Asp(85)) similar or equal to 7.2 was a lso employed. A major objective was to verify whether the basic relati onships of homogeneous kinetics obeyed by elementary acid/base systems in solution (primarily, the possibility to express the equilibrium co nstant as the ratio of the forward and back rate constants) are also o beyed by the Schiff base and Asp(85) moieties. We found that this is t he 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 ba se is titrable from the outside medium via a proton channel, which bec omes saturated, and thus rate determining, below pH congruent to 7. Th e 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 order s of magnitude as compared to the diffusion-controlled rate constant o f an elementary acid/base in homogeneous solutions. In the case of Asp (85), ka could not be directly determined. The titration rates observe d in the case of pigment IV are, however, consistent with a model in w hich the Schiff base and Asp(85) are exposed to the extracellular side via the same proton channel. It is suggested that the rate-determinin g step in proton translocation via this channel is a transfer between Asp(85) and the outside, rather than between Asp(85) and the Schiff ba se. This conclusion applies independently of whether Asp(85) is proton ated or non-protonated. The results are relevant to basic questions re lated to the proton pump mechanism in bR, primarily (a) the exposure d irection (to the outside or to the inside of the cell) of the Schiff b ase 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 i s highly exposed to the outside medium. More generally, our study bear s on the basic problem concerning the relationship between the kinetic s of the titration of protein residues and their respective (''thermod ynamic'') equilibrium constants.