Photoreaction of bacteriorhodopsin at high pH: origins of the slow decay component of M

Abstract

The absorption spectrum of light-adapted purple membrane in 3 M KCl is dependent on temperature even in the room temperature region. Temperature-induced difference spectra at various pH values suggested that the trans isomer of bacteriorhodopsin, bR570, is in thermal and/or photodynamic equilibrium with several different conformers. The major second conformer occurring at neutral pH had the same spectroscopic properties as the 13-cis isomer, and its content at 35 degrees C was estimated to be more than 20%. Heterogeneity in the protein conformation became more significant above pH8, where temperature-induced difference spectra exhibited a negative peak at 580 nm and a positive peak at 296 nm. This absorption change is very similar to that observed upon the formation of the N intermediate, suggesting that an N-like conformer occurs at high pH and temperature. A significant temperature dependence was also seen in the M decay kinetics at high pH, which were described by two decay components; i.e., the fast decaying M (Mf) was predominant at low temperature, but the amplitude of the slow component (M(s)) increased with increasing temperature. It is suggested that M(s) is generated upon excitation of the N-like conformer, in which the residue (Asp-96) usually acting as a proton donor to the Schiff base is deprotonated. The N-like conformer could be N itself, because M(s) was enhanced when N was accumulated by background light. A strong correlation between the amplitude of M(s) and the concentration of N was also revealed by the accumulation kinetics of Mf, M(s), and N after the onset of continuous actinic light.(ABSTRACT TRUNCATED AT 250 WORDS)

Proton release pathway in bacteriorhodopsin: Molecular dynamics and electrostatic calculations

“…The motion of R82 during the photocycle was proposed earlier [ 10,17,54]. Large protein conformational changes during the lifetime of M were found experimentally [59]. The strong pH dependence of the M2 electrogenicity indicates [65] that the MI to M2 transition involves complex charge motions, as is expected in the proposed conformational change of the protein.…”

“…There are at least three additional states: a blue shifted ( A, , , 480 nm) all-trans component, a N-like species possibly with deprotonated D96 [59] and a red-shifted species which is attributed to the ionization of a tyrosine residue [60]. Two different BR states ( a , p in a pH-dependent population ratio)…”

Interconversions among four M‐intermediates in the bacteriorhodopsin photocycle

“…The pH dependence of the BR photocycle is quite complex and is influenced by ionic strength and other assay conditions as well as the time domain studied and parameters that are measured [12–16]. In our study, a limited pH range which encompassed the titrations of both Mf and Ms was used.…”

Effect of Xenon Binding to a Hydrophobic Cavity on the Proton Pumping Cycle in Bacteriorhodopsin

“…The kinetic model of Chizhov et al 39 was used for data analysis. 41 With the use of the singular-value decomposition method, 64 absorption kinetics measured at various wavelengths were decomposed into four major singular-value vectors, from which the optimal number of exponential components and individual decay constants were determined by use of the program Origin 7.5. The nonlinear leastsquares method was then applied to find a single set of exponential functions by which the absorption change at each wavelength is best fitted.…”