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 b ound to their apoproteins via a protonated Schiff base. We have employ ed continuous-flow resonance Raman experiments on both pigments to det ermine that the exchange of a deuteron on the Schiff base with a proto n 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., a cid-, base-, or water-catalyzed schemes, it is found that none of thes e can explain the experimental results. Because the exchange rates are found to be independent of pH, the deuterium-hydrogen exchange can no t be hydroxyl (or acid-)catalyzed. Moreover, the deuterium-hydrogen ex change of the retinal Schiff base cannot be catalyzed by water acting as a base because in that case the estimated exchange rate is predicte d to be orders of magnitude slower than that observed. The relatively slow calculated exchange rates are essentially due to the high pKa val ues of the Schiff base in both rhodopsin (pKa > 17) and bacteriorhodop sin (pKa similar to 13.5). We have also measured the deuterium-hydroge n exchange of a protonated Schiff base model compound in aqueous solut ion. Its exchange characteristics, in contrast to the Schiff bases of the pigments, is pH-dependent and consistent with the standard base-ca talyzed 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 slowerthan the exchange rate of the Schiff base in rhodopsin and bacte riorhodopsin. 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 protei ns over the model Schiff base in aqueous solution. We suggest that the se results are explained by the presence of a structural water molecul e (or molecules) at the retinal binding sites of the two pigments, qui te close, probably-hydrogen bonded, to the Schiff base proton. In this case, the rate of exchange can be faster than that found for the mode l compound due to an ''effective water concentration'' near the Schiff base that is increased from that found in aqueous solution.