TRP86-]PHE REPLACEMENT IN BACTERIORHODOPSIN AFFECTS A WATER MOLECULE NEAR ASP85 AND LIGHT ADAPTATION

Illumination of the Trp86 --> Phe mutant of bacteriorhodopsin causes a nomalous light adaptation, i.e., isomerization of the retinal from all -trans to 13-cis, 15-syn. FTIR spectral analysis shows that illuminati on at 250 K yields two 13-cis photoproducts, the conventional 13-cis, 15-syn state, BRC, and another termed BRX. BRX is different from BRC b ecause it has a lower N-H in-plane bending frequency and a higher C-14 -C-15 stretching frequency, as well as an absence of coupling between these modes. BRX, which is stable at 275 K, is more abundant in the ph otosteady state produced by longer wavelength light and detected as th e only photoproduct at 170 K. Its different structural features result from distortion of the C-14-C-15 bond of the chromophore. In the W86F mutant protein, the small structural changes of a water molecule in t he conversion between the all-trans and 13-cis, 15-syn forms and in th e formation of the K photointermediate are absent, but the larger chan ges of water molecule(s) that normally occur in the L and M intermedia tes are present. We propose that Trp86, together with Asp85, is involv ed in binding the water molecule and in preventing the formation of th e 13-cis, 15-syn photoproducts, BRC and BRX, when the wild type protei n is illuminated.