Evidence for a bound water molecule next to the retinal Schiff base in bacteriorhodopsin and rhodopsin: a resonance Raman study of the Schiff base hydrogen/deuterium exchange.
The retinal chromophores of both rhodopsin and bacteriorhodopsin are bound to their apoproteins via a protonated Schiff base. We have employed continuous-flow resonance Raman experiments on both pigments to determine that the exchange of a deuteron on the Schiff base with a proton is very fast, with half-times of 6.9 +/- 0.9 and 1.3 +/- 0.3 ms for rhodopsin and bacteriorhodopsin, respectively. When these results are analyzed using standard hydrogen-deuteron exchange mechanisms, i.e., acid-, base-, or water-catalyzed schemes, it is found that none of these can explain the experimental results. Because the exchange rates are found to be independent of pH, the deuterium-hydrogen exchange can not be hydroxyl (or acid-)-catalyzed. Moreover, the deuterium-hydrogen exchange of the retinal Schiff base cannot be catalyzed by water acting as a base because in that case the estimated exchange rate is predicted to be orders of magnitude slower than that observed. The relatively slow calculated exchange rates are essentially due to the high pKa values of the Schiff base in both rhodopsin (pKa > 17) and bacteriorhodopsin (pKa approximately 13.5). We have also measured the deuterium-hydrogen exchange of a protonated Schiff base model compound in aqueous solution. Its exchange characteristics, in contrast to the Schiff bases of the pigments, is pH-dependent and consistent with the standard base-catalyzed schemes. Remarkably, the water-catalyzed exchange, which has a half-time of 16 +/- 2 ms and which dominates at pH 3.0 and below, is slower than the exchange rate of the Schiff base in rhodopsin and bacteriorhodopsin. Thus, there are two anomalous results, the inconsistency of the observed hydrogen exchange rates of retinal Schiff base in the two pigments with those predicted from the standard exchange schemes and the enhancement of the rate of hydrogen exchange in the two proteins over the model Schiff base in aqueous solution. We suggest that these results are explained by the presence of a structural water molecule (or molecules) at the retinal binding sites of the two pigments, quite close, probably-hydrogen bonded, to the Schiff base proton. In this case, the rate of exchange can be faster than that found for the model compound due to an "effective water concentration" near the Schiff base that is increased from that found in aqueous solution.
Department of Physics, City College of the City University of New York, New York 10031.
H
H
Deng
L
L
Huang
R
R
Callender
T
T
Ebrey
D001436:Bacteriorhodopsins / Q000737:chemistry*; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D003903:Deuterium; D006859:Hydrogen; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008956:Models, Chemical; D011522:Protons; D012172:Retinaldehyde / Q000737:chemistry; D012243:Rhodopsin / Q000737:chemistry*; D012545:Schiff Bases / Q000737:chemistry; D013059:Spectrum Analysis, Raman; D014867:Water / Q000737:chemistry
D011522:Protons; D012545:Schiff Bases; D014867:Water; D001436:Bacteriorhodopsins; D006859:Hydrogen; D012243:Rhodopsin; D003903:Deuterium; D012172:Retinaldehyde