Photocycle and Vectorial Proton Transfer in a Rhodopsin from the Eukaryote <i>Oxyrrhis marina</i>

Retinylidene photoreceptors are ubiquitously present in marine protists as first documented by the identification of green proteorhodopsin (GPR). We present a detailed investigation of a rhodopsin from the protist <i>Oxyrrhis marina</i> (OR1) with respect to its spectroscopic properties and to its vectorial proton transport. Despite its homology to GPR, OR1’s features differ markedly in its pH dependence. Protonation of the proton acceptor starts at pH below 4 and is sensitive to the ionic conditions. The mutation of a conserved histidine H62 did not influence the p<i>K</i>_a value in a similar manner as in other proteorhodopsins where the charged histidine interacts with the proton acceptor forming the so-called His-Asp cluster. Mutational and pH-induced effects were further reflected in the temporal behavior upon light excitation ranging from femtoseconds to seconds. The primary photodynamics exhibits a high sensitivity to the environment of the proton acceptor D100 that are correlated to the different initial states. The mutation of the H62 does not affect photoisomerization at neutral pH. This is in agreement with NMR data indicating the absence of the His-Asp cluster. The subsequent steps in the photocycle revealed protonation reactions at the Schiff base coupled to proton pumping even at low pH. The main electrogenic steps are associated with the reprotonation of the Schiff base and internal proton donor. Hence, OR1 shows a different theme of the His-Asp organization where the low p<i>K</i>_a of the proton acceptor is not dominated by this interaction, but by other electrostatic factors

Pre-Gating Conformational Changes in the ChETA Variant of Channelrhodopsin‑2 Monitored by Nanosecond IR Spectroscopy

Light-gated ion permeation by channelrhodopsin-2 (ChR2) relies on the photoisomerization of the retinal chromophore and the subsequent photocycle, leading to the formation (on-gating) and decay (off-gating) of the conductive state. Here, we have analyzed the photocycle of a fast-cycling ChR2 variant (E123T mutation, also known as ChETA), by time-resolved UV/vis, step-scan FT-IR, and tunable quantum cascade laser IR spectroscopies with nanosecond resolution. Pre-gating conformational changes rise with a half-life of 200 ns, silent to UV/vis but detected by IR spectroscopy. They involve changes in the peptide backbone and in the H-bond of the side chain of the critical residue D156. Thus, the P₁⁵⁰⁰ intermediate must be separated into early and late states. Light-adapted ChR2 contains a mixture of all-<i>trans</i> and 13-<i>cis</i> retinal in a 70:30 ratio which are both photoactive. Analysis of ethylenic and fingerprint vibrations of retinal provides evidence that the 13-<i>cis</i> photocycle recovers in 1 ms. This recovery is faster than channel off-gating and most of the proton transfer reactions, implying that the 13-<i>cis</i> photocycle is of minor functional relevance for ChR2

Phylogenetic tree inferred using the Maximum likelihood method of 21 amino acid sequences of <i>Exiguobacterium</i> spp. closely related with E17R.

<p>The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.</p

Absorption spectrum of E17R in 20 mM Hepes, 100 mM NaCl, 0.03% DDM.

<p>The inset shows the difference spectrum after bleaching with hydroxylamine. The extinction coefficient was calculated using the absorbance of the oxime product ΔA(oxime) in comparison to the bleached rhodopsin absorbance ΔA(Rh) as reference.</p

Multiple protein alignment of PR from <i>Exiguobacterium</i> sp. S17 (E17R), green-light absorbing proteorhodopsin from <i>Exiguobacterium sibiricum</i> (ESR) and blue-light absorbing proteorhodopsin from the uncultured gamma-proteobacterium “Hot 75m4” (BPR).

<p>Residues shared between all the protein variants are marked with asterisks. Single amino acid residue at position 106 (BPR numbering) that functions as a spectral tuning switch and accounts for most of the spectral difference between the two pigment families is highlighted in light grey. Primary proton acceptor and donor are highlighted in dark grey (D86) and with a frame (K97), respectively. The Schiff base (K232 for ESR, K226 for E17R) is indicated by a diamond. Residues differing between both green-PRs are indicated with arrows. The seven transmembrane α-helices are indicated with blue bubbles.</p

Transient absorbance changes of E17R.

<p>A. The kinetics of the absorbance changes are shown for selected wavelengths (399 nm, 517 nm and 598 nm). B. The decay-associated spectra are depicted as obtained from the global fit. The corresponding time constants are given in the figure.</p

Electrometric record of E17R (in 20 mM Hepes, pH 7.4) with the BLM system.

<p>A. Transient currents and B. after the addition of the protonophore FCCP. Black bars indicate illumination with a 75 W XBO long-pass filtered at >495 nm. The grey bar shows the additional excitation of the M-state (>380 nm).</p