Evidence for the involvement of more than one metal cation in the Schiff base deprotonation process during the photocycle of bacteriorhodopsin

Abstract

The removal of metal cations inhibits the deprotonation process of the protonated Schiff base during the photocycle of bacteriorhodopsin. To understand the nature of the involvement of these cations, a spectroscopic and kinetic study was carried out on bacteriorhodopsin samples in which the native Ca2' and Mg2e were replaced by Eu3+, a luminescent cation. The decay of Eu3' emission in bacteriorhodopsin can be fitted to a minimum of three decay components, which are assigned to Eu31 emission from three different sites. This is supported by the response of the decay components to the presence of 2H20 and to the changes in the Eu3+/bR molar ratio. The number of water molecules coordinated to Eu3' in each site is determined from the change in its emission lifetime when 2H20 replaces H20. Most of the emission originates from two "wet" sites of low crystal-field symmetry--e.g., surface sites. Protonated Schiff base deprotonation has no discernable effect on the emission decay of protein-bound Eu3+, suggesting an indirect involvement of metal cations in the deprotonation process. Adding Eu3+ to deionized bacteriorhodopsin increases the emission intensity of each Eu3+ site linearly, but the extent of the deprotonation (and color) changes sigmoidally. This suggests that if only the emitting Eu3+ ions cause the deprotonation and bacteriorhodopsin color change, ions in more than one site must be involved-e.g., by inducing protein conformation changes. The latter could allow deprotonation by the interaction between the protonated Schiff base and a positive field of cations either on the surface or within the protein.Bacteriorhodopsin (bR) is the only protein found in the purple membrane of Halobacterium halobium, a light-harvesting bacterium. It contains a single chromophore molecule, retinal, covalently bound via a protonated Schiff base (PSB) linkage to the E-amino group of a lysine residue in the protein (1, 2). Upon absorbing a photon, it undergoes a photochemical cycle (3) consisting of at least four intermediates on time scales varying from pico-to milliseconds: bR-7* K610 L55 -M412 06 O --, bR570.During this photocycle, protons are pumped across the cell membrane to the outside, establishing an electrochemical proton gradient used by the organism for metabolic processes such as ATP synthesis (ref. 4 and refs. therein). Protons are ejected from the cell at a rate comparable to the formation of the M412 intermediate (5,6). A good correlation has been found between the number of protons pumped and the amount of the slow-decaying form of M412 (7). This intermediate is the only one in which the Schiff base is unprotonated (8, 9). Consequently, many studies have inferred that the PSB deprotonation is closely associated with the proton pump mechanism (for review see ref. 6).Recently, a very simple electrostatic model (the cation model) was proposed (10) to account for the strong coupling between (10-14), and thus the similar mechanism of deprotonation of, the PSB and an acid with a pKa value of 8.6-10 [possibly tyro...

Modification of two peptides of bacteriorhodopsin with a (pentaammine)cobalt(III) complex

“…However, the properties of the cation binding site described here might be similar to those of the hydrophobic site proposed by Corcoran et al (1987).…”

“…Ariki et al (1987) concluded from the lanthanide luminescence of Eu3+-modified bR that two high-affinity sites with six protein ligands were present. In a similar study, Corcoran et al (1987) determined three different cation environments, two of which are surface sites and with six and three water molecules, respectively, as ligands. The cation in the third site is probably located in a hydrophobic region.…”

Tryptophan fluorescence quenching as a monitor for the protein conformation changes occurring during the photocycle of bacteriorhodopsin under different perturbations.

“…The deprotonation probability is found (11,15,19) to have a nonlinear dependence on the metal cation concentration and to have no effect on the metal (Eu3+) cation binding sites. This suggests an indirect (20,21) involvement of metal cations in the deprotonation process or blue-to-purple color transition-e.g., by controlling the protein conformation changes during the cycle (22). "Acid purple" bR (bR at pH 0) is found to form K610 and L550 analogues (15-17) but does not form M412 or pump protons (15)(16)(17).…”

PURPLE MEMBRANE: SURFACE CHARGE DENSITY and THE MULTIPLE EFFECT OF pH and CATIONS

“…A further investigation of the location of a cation bound in this region is currently under investigation (Engelhard et al, 1990;abstract). Corcoran et al (1987) replaced the endogenous divalent cations in the purple membrane with Eu3+ because the emission decay components could be used to estimate the number of emitting sites and the degree of hydration of the ions. Although four strongly binding Eu3+ sites were determined by flame atomic emission spectrophotometry, only three sites were observed by monitoring the luminescence.…”