Specific tryptophan UV-absorbance changes are probes of the transition of rhodopsin to its active state

The difference of rhodopsin and metarhodopsin II (MII) absorption spectra exhibits a characteristic pattern in the UV wavelength range, consisting of peaks at 278, 286, 294, and 302 nm. These difference bands are thought to result from the perturbation of the environments of tryptophan and/or tyrosine residues, We used site-directed mutagenesis to investigate the contribution of tryptophan absorption to these spectral features, Each of the five tryptophan residues in bovine rhodopsin was replaced by either a phenylalanine or a tyrosine. The mutant pigments (W35F, W126F, W161F, W175F, W265F/Y) were prepared and studied by UV-visible photobleaching difference spectroscopy, The difference spectra of the W35F and W175F mutants were identical to that of rhodopsin, whereas in the W161F mutant, the magnitudes of the 294- and 302-nm bands were slightly lowered, The differential absorbance at 294 nm was reduced by over 50% in the W126F and W265F/Y mutants, The difference peak at 302 nm was reduced in the W265F/Y mutants, but was almost completely absent in the W126F mutant, These data indicate that the difference bands at 294 and 302 nm originate from the perturbations of Trp(126) and Trp(265) environments resulting from a general conformational change concomitant with MII formation and receptor activation, Model studies on tryptophan absorption indicate that the difference peak at 294 nm is due to the differential shift of the L(b) absorption of the indole side chain as a result of decreased hydrophobicity or polarizability of the Trp(126) and Trp(265) environments. The resolution of the 302-nm band, assigned to the differential shift of the indole L(a) absorption, is consistent with hydrogen-bonding interactions of the indole N-H groups of Trp(126) and Trp(265) becoming weaker in MTI. These results suggest that the photoactivation of rhodopsin involves a change in the relative disposition of transmembrane helices 3 and 6, which contain Trp(126) and Trp(265) respectively, within the alpha-helical bundle of the receptor.