Light-dependent trans to cis isomerization of the retinal in halorhodopsin

Flash-induced absorption changes in the near UV were determined for bacteriorhodopsin and halorhodopsin on a millisecond time scale. The difference spectrum obtained for bacteriorhodopsin was comparable to model difference spectra of tyrosine (aromatic OH deprotonated vs protonated), as found by others. The flash-induced difference spectrum for halorhodopsin, in contrast, resembled a model spectrum obtained for trans to 13-cis isomerization of retinal in bacteriorhodopsin. A model for chloride translocation by halorhodopsin is presented, in which the retinal isomerization moves positive charges, which in turn modulate the affinity of a site to chloride.     

Characterization of the Photochemical Reaction Cycle of Proteorhodopsin

Absorption changes in the photocycle of the recently described retinal protein, proteorhodopsin, are analyzed. The transient spectra at pH 9.5, where it acts as a light-driven proton pump, reveal the existence of three spectrally different intermediates, K, M, and N, named in analogy with the photointermediates of bacteriorhodopsin. Model analysis based on time-dependent absorption kinetic signals at four wavelengths suggested the existence of two more spectrally silent intermediates and lead to a sequential reaction scheme with five intermediates, K, M₁, M₂, N, and PR′, before decay to the initial state PR. An L-like intermediate was not observed, probably for kinetic reasons. By measuring the light-generated electric signal of an oriented sample, the electrogenicity of each intermediate could be determined. The electrogenicities of the first three intermediates (K, M₁, and M₂) have small negative value, but the last three components, corresponding to the N and PR′ intermediates and PR, are positive and two-orders-of-magnitude larger. These states give the major contributions to the proton translocation across the membrane. The energetic scheme of the photocycle was calculated from the temperature-dependence of the absorption kinetic signals.     

X-Ray Crystallography of Bacteriorhodopsin and Its Photointermediates: Insights into the Mechanism of Proton Transport

In the last few years, detailed structural information from high-resolution x-ray diffraction has been added to the already large body of spectroscopic and mutational data on the bacteriorhodopsin proton transport cycle. Although there are still many gaps, it is now possible to reconstruct the main events in the translocation of the proton and how they are coupled to the photoisomerization of the retinal chromophore. Future structural work will concentrate on describing the details of the individual proton transfer steps during the photocycle.