Cis–Trans Reisomerization Preceding Reprotonation of the Retinal Chromophore Is Common to the Schizorhodopsin Family: A Simple and Rational Mechanism for Inward Proton Pumping

Abstract

The creation of unidirectional ion transporters across membranes represents one of the greatest challenges in chemistry. Proton-pumping rhodopsins are composed of seven transmembrane helices with a retinal chromophore bound to a lysine side chain via a Schiff base linkage and provide valuable insights for designing such transporters. What makes these transporters particularly intriguing is the discovery of both outward and inward proton-pumping rhodopsins. Surprisingly, despite sharing identical overall structures and membrane topologies, these proteins facilitate proton transport in opposite directions, implying an underlying rational mechanism that can transport protons in different directions within similar protein structures. In this study, we unraveled this mechanism by examining the chromophore structures of deprotonated intermediates in schizorhodopsins, a recently discovered subfamily of inward protonpumping rhodopsins, using time-resolved resonance Raman spectroscopy. The photocycle of schizorhodopsins revealed the cis− trans thermal isomerization that precedes reprotonation at the Schiff base of the retinal chromophore. Notably, this order has not been observed in other proton-pumping rhodopsins, but here, it was observed in all seven schizorhodopsins studied across the archaeal domain, strongly suggesting that cis−trans thermal isomerization preceding reprotonation is a universal feature of the schizorhodopsin family. Based on these findings, we propose a structural basis for the remarkable order of events crucial for facilitating inward proton transport. The mechanism underlying inward proton transport by schizorhodopsins is straightforward and rational. The insights obtained from this study hold great promise for the design of transmembrane unidirectional ion transporters.

Chromophore–Protein Interactions Affecting the Polyene Twist and π–π* Energy Gap of the Retinal Chromophore in Schizorhodopsins

“…This nonuniformity is further strong evidence that the distortion of the polyene chain is caused not by the hydrogen bond between the Schiff base and the Asp residue but by other interactions with nearby residues. We recently proposed that tight atomic contacts between the C 13 C 14 group and nearby residues (Trp154 and Tyr71 in SzR4) facilitate the cis – trans thermal isomerization of SzRs . Such tight contacts can distort the polyene chain near the Schiff base since protein structures are generally compact …”

“…The distortion of the retinal polyene chain can affect the barrier height for structural changes in the chromophore and thus their rate of change. We previously revealed that cis – trans re-isomerization precedes re-protonation in the photocycle of SzRs. , Therefore, we previously discussed the correlation between the rate of cis – trans re-isomerization and distortion of the retinal polyene chain. Figure shows plots of the re-isomerization rate against the HOOP intensities.…”

“…Collectively, these results indicate that the re-isomerization rate increases as the atomic contacts become tighter. This is consistent with our previous proposal that tight contacts distort the polyene chain and thus facilitate re-isomerization by lowering its barrier height …”

“…M sSzR, Mt SzR, and AntR were expressed and purified by methods reported previously Escherichia coli C43­(DE3) (Lucigen) containing a His-tagged SzR plasmid was cultivated in 2 × YT at 37 °C.…”

“…Unexpectedly, polyene distortion decreased as the hydrogen bond became stronger in the SzRs, suggesting that the distortion is due to tight atomic contacts between the chromophore and nearby residues. The positive correlation between the degree of distortion and the cis – trans thermal isomerization rate is consistent with our previous proposal that the tight contacts facilitate isomerization . The present study revealed the effects of the tight contacts and the hydrogen bond of the Schiff base on the chromophore properties in SzRs, thereby shedding light on potential modifications to the properties of retinal proteins.…”