Effects of tyrosine-26 and tyrosine-64 nitration on the photoreactions of bacteriorhodopsin

Abstract

In the dark, all titratable tyrosine residues of bacteriorhodopsin have pK's of greater than 11.0, which may be caused by the hydrophobic environment for buried residues and by high negative charge density for surface residues [Scherrer, P., & Stoeckenius, W. (1984) Biochemistry 23, 6195-6202]. Under illumination, deprotonation of only one tyrosine is observed in the micro- and millisecond time ranges of the photocycle; this is Tyr-64. Nitration of Tyr-64 decreases the chromophore absorbance, shifts the absorption maximum to 535 nm, and affects photocycle kinetics. However, restoring its native pK by reduction after nitration has no effect on the changes in photocycle kinetics or absorbance of the chromophore. Nitration of Tyr-64 apparently causes a conformational change in bR, which is independent of the pK of its phenolic group. These observations contradict earlier conclusions that in the photocycle a tyrosine residue directly interacts with the Schiff base during its deprotonation or reprotonation. The protonation state of Tyr-26 and the alkaline chromophore transition are correlated, as shown earlier (Scherrer & Stoeckenius, 1984). Lowering the pK of Tyr-26 by nitration decreases the M-decay rate, and this effect is partially reversed by reduction of the nitro group. We conclude that Tyr-26 may be located close to the chromophore and interact with it; but its protonation state does not change at physiological pH and in the microsecond time range of the photocycle. Tyr-64 is apparently located at or close to the external surface; its modification strongly affects the chromophore but apparently indirectly and not through its protonation changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriorhodopsin mutants containing single tyrosine to phenylalanine substitutions are all active in proton translocation.

“…Further, protonation and deprotonation of at least two tyrosine residues during the photocycle have been suggested from Fourier-transform infrared spectroscopy (8,9) and kinetic and low-temperature UV spectroscopy (10)(11)(12). Chemical modification studies also implicated Tyr-26 and Tyr-64 in proton-pumping mechanism (13,14), although these conclusions were at variance with later work (15).…”

Picosecond Multidimensional Fluorescence Spectroscopy: A Tool to Measure Real-time Protein Dynamics During Function†

“…2). As the formation of the O‐intermediate is favored at higher temperatures and acidic pH‐values (29), the amplitude of the 5‐ms decay component at 10°C and pH 7 amounts only to 16% of the total amplitude, consistent with a 10–30% accumulation of the O‐intermediate at 10°C. A closer look at the amplitudes of the fluorescence intensity change kinetics at 166 ps after fluorescence excitation (Fig.…”

Vibrational Spectra of Rhodopsin and Bacteriorhodopsin

“…In addition to these new intermediates, the presence of multiple forms was repeatedly suggested for M412 from the biphasic nature of its decay (Stoeckenius and Bogomolni, 1982;Ohno et al, 1981;Li et al, 1984;Scherrer and Stoeckenius, 1985;Groma and Dancshazy, 1986;Grzesiek and Dencher, 1986) and also for its formation (Hanamoto et al, 1984). Furthermore, a very fast-decaying M412 species with the decay constant of 0.39 ms was found from transient Raman spectra of weakly alkaline solutions (Nakagawa et al, 1989b).…”