Bovine rhodopsin contains 11-cis-retinal as a light-absorbing chromophore that binds to a lysine residue of the apoprotein opsin via a protonated Schiff base linkage. Light isomerizes 11-cis-retinal into the all-trans form, which eventually leads to the formation of an enzymatically active state,metarhodopsin II (MII). It is widely believed that MII forms a pH-dependent equilibriumwithmetarhodopsin I (MI), but direct evidence for this equilibrium has not been reported. Here, we confirmed this equilibrium by direct observation of the mutual conversions ofMI andMII upon changing the pH of theMI/MIImixture.We also observed a reversible binding of the synthetic peptide constituting the C-terminal 11 amino acids of the transducin α-subunit to MII, which resulted in change of the amounts of MI and MII in the equilibrium. Interestingly, addition of the peptide did not induce a simple pKa shift but rather induced an increase of the MII fraction at high pH. These results indicate that in addition to theMII that is formed from MI in a pH-dependentmanner there also exists another MII, which is in equilibriumwith MI in a pH-independentmanner and can bind to the peptide. Therefore, there is no need for proton uptake by the protein moiety of opsin for the binding to the peptide.
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