Contact-Mediated Retinal–Opsin Coupling Enables Proton Pumping in Gloeobacter Rhodopsin

Abstract

When a chromophore embedded in a photoreceptive protein undergoes a reaction upon photoexcitation, the photoreaction triggers structural changes in the protein moiety that are necessary for the function of the protein. It is thus essential to elucidate the coupling between the chromophore and protein moiety to understand the functional mechanism for photoreceptive proteins, but the mechanism by which this coupling occurs remains poorly understood. Here, we show that nonbonded atomic contacts play an essential role in driving functionally important structural changes following photoisomerization of the chromophore in Gloeobacter rhodopsin (GR). Time-resolved ultraviolet resonance Raman spectroscopy revealed that the substitution of Trp222, which contacts with methyl groups of the retinal chromophore, with a Phe residue reduced the extent of structural change. The proton-pumping activity of the GR mutant was as small as 9% of that of the wild type. Time-resolved visible absorption and resonance Raman spectra showed that the photocycle of the mutant proceeded to the L intermediate following the all-trans to 13-cis photoisomerization step but did not result in the deprotonation of the chromophore. The present results demonstrate that the atomic contacts between the chromophore and the Trp222 side chain induce the structural changes necessary for proton transfer. The requirement for dense atomic packing in a protein structure for the efficient propagation of structural changes through a coupling mechanism is discussed.

Origin of a Double-Band Feature in the Ethylenic C═C Stretching Modes of the Retinal Chromophore in Heliorhodopsins

“…40,41 We recently revealed that atomic contacts between the Trp side chain and the 13-methyl group in the 13-cis form drive protein structural changes necessary for intraprotein proton transfer in Gloeobacter rhodopsin. 42 In contrast, in HeRs, it is likely that steric contacts of the 13-methyl group with the Phe side chain in the 13-cis form are weak because the Phe side chain is shifted toward the ionone ring side compared to that in type-1 rhodopsins. Therefore, we hypothesize that, in HeRs, the reduced contacts of the 13-methyl group decelerate thermal cis−trans isomerization and increase the lifetime of the last intermediate in the photocycle.…”

“…Our time-resolved resonance Raman study revealed that the long-lived intermediate of HeR 48C12 adopted the 13- cis configuration, implying that cis – trans isomerization occurred during the transition from the O intermediate to the unphotolyzed state. In Hs BR, the 13-methyl group in the 13- cis form was shown to conflict with the Trp side chain. , We recently revealed that atomic contacts between the Trp side chain and the 13-methyl group in the 13- cis form drive protein structural changes necessary for intraprotein proton transfer in Gloeobacter rhodopsin . In contrast, in HeRs, it is likely that steric contacts of the 13-methyl group with the Phe side chain in the 13- cis form are weak because the Phe side chain is shifted toward the ionone ring side compared to that in type-1 rhodopsins.…”

Proton Release Reactions in the Inward H+ Pump NsXeR

“…Together with reorientations of L93 and F219, those structural changes were shown to create space for a linear water chain connecting D96 to the RSB, in line with crystallographic data . It was shown for another outward proton pump, Gloeobacter rhodopsin (GR), that the steric repulsion between retinal and the homologous W222 is functionally highly relevant, as M state formation is hindered in the W222F variant . In Ns XeR, amide I bands at 1658 (−) and 1670 (+) are already present in MI and increase in intensity during the MI/MII transition, where proton transfer to D220 occurs.…”

Protein dynamics of a light-driven Na⁺ pump rhodopsin probed using a tryptophan residue near the retinal chromophore

“…We examined wild-type (WT) KR2 and the W215F mutant, in which Trp215, widely conserved in microbial rhodopsins [2], is replaced by phenylalanine (Phe). Trp215 is located on the cytoplasmic side near the retinal chromophore (Figure 1) and is believed to be a mechanical transducer during function, as demonstrated for other ion-pumping rhodopsins [2,[29][30][31]. Ion pump activity and transient absorption spectroscopy indicated that the atomic contact between the bulky side chain of Trp215 and the C20 methyl group of the retinal facilitates relaxation from the O intermediate to the unphotolyzed state.…”