Movement of the retinylidene Schiff base counterion in rhodopsin by one helix turn reverses the pH dependence of the metarhodopsin I to metarhodopsin II transition.

The environment of the retinylidene Schiff base in bovine rhodopsin has been studied by movement of its carboxylic acid counterion from position 113 to position 117 by site-specific mutagenesis. Replacement of the counterion at position 113 by a neutral amino acid residue has been shown to produce a lowering of the Schiff base acidity constant (pKa) from > 8.5 to about 6. The aim of the present work was to change the position of the counterion without causing a significant effect on the Schiff base pKa. A triple replacement mutant (Glu113-->Ala/Ala117-->Glu/Glu122-->Gln) was designed to move the position of the counterion by one helix turn in the third putative transmembrane helix (helix C). The mutant bound 11-cis-retinal to form a chromophore with a visible absorbance maximum (lambda max) of 490 nm which was independent of pH in the range of about 5-8.5. Upon illumination under conditions in which rhodopsin was converted to the active metarhodopsin II (MII) photoproduct, the mutant was converted to a metarhodopsin I (MI)-like species (lambda max = 475 nm). Furthermore, the effect of pH on the photobleaching behavior of the mutant was the reverse of that reported for rhodopsin. In the mutant, acidic pH favored the formation of the MI-like photoproduct, and basic pH favored the formation of an MII-like photoproduct (lambda max = 380 nm). The MII-like photoproduct of the mutant pigment was able to activate the guanine nucleotide-binding protein, transducin. We conclude that the Schiff base counterion in rhodopsin can be repositioned to form a pigment with an apparently unperturbed Schiff base pKa. Furthermore, a specific amino acid residue that acts as a Schiff base proton acceptor is not strictly required for photoconversion of rhodopsin to its active MII form.