BATHOPRODUCTS and CONFORMERS OF ALL‐trans‐AND 13‐ds BACTERIORHODOPSIN AT 90 K*

Abstract

At 90 K the photoproduct of the primary light reaction of trans-bacteriorhodopsin, the bathoproduct K', consists of a mixture of at least three spectrally different species, K;, K;, and K;,,, having maxima i n the difference absorption spectra at 645, 635 and 625 nm, respectivcly. The bathoproducts differ in their long wavelength absorption bands and in their rate constants for photoconversion to tram-bacteriorhodopsin under far red light irradiation (A > 720 nm). The bathoproducts are formed from different precursors+onformers of trans-bacteriorhodopsin, which are stable at 90 K, but are in equilibrium with each other at 213 K. We suggest that the bathoproducts may initiate parallel conversion cycles of trans-bacteriorhodopsin at low temperatures. The primary photoreaction of 13-cis-bacteriorhodopsin also yields three bathoproducts, K;, K;, and K;,,, having maxima in the difference absorption spectra at 615, 605 and 595 nm, respectively.

pH dependence of light‐induced proton release by bacteriorhodopsin

“…This result suggests an interaction between Arg-82 and tyrosine(s) during the L to M intermediate transition, consistent with our results here and our studies on Arg-82 and Tyr-57 mutants and spectral titrations [ 19,20,26]. Under alkaline conditions, a negatively charged tyrosinate near would neutralize the charge on Arg-82, resulting in an environment similar to that in the Arg-82 mutant.…”

“…The intrinsic pK, that we determined for the loss of normal proton release correlates with the previously found titration of a tyrosine residue [19]. The removal of the positively charged Arg-82 results in behavior similar to that of the alkaline form [26].…”

“…The role of Arg-82 is intriguing because substitution of this positively charged residue with a neutral one (Ala) makes the charge in the active site more negative and accelerates the formation rate of the M intermediate similar to that of the alkaline form of the native bacteriorhodopsin (which we believe is attributed to tyrosine deprotonation [19]) termediate of the Arg-82 Asn mutant was much faster than that of the alkaline form; however. the decay rate is more likely controlled by the protonation state of Asp-96 located on the cytoplasmic half of the protein.…”

“…At high pH, a tyrosine in bacteriorhodopsin has been proposed to be able to deprotonate with an intrinsic pK of about 8.3 resulting in a red-shifted form of bR (bR a,kd,,ne), which undergoes a photocycle different from that at neutral pH ( Fig. 1) [19]. Govindjee et al [20] report spectral titrations at 240 nm showing that this low pK, tyrosine a bacteriorhodopsin is missing in a mutant in which Tyr-57 is replaced with Asn.…”

Bacteriorhodopsin photocycle at cryogenic temperatures reveals distributed barriers of conformational substates

“…Spectral and kinetic anomalies near 100 K had suggested the existence of multiple K photoproducts (16,17), which come to a steady-state mixture only at temperatures above 200 K (16), in a suggested analogy with myoglobin. Similar findings were reported for the low-temperature bathoproduct of 13-cis bacteriorhodopsin (17). In a different approach, optical hole burning (18) indicated the existence of vibrational substates that give rise to inhomogeneous band broadening in this protein.…”