His-75 in Proteorhodopsin, a Novel Component in Light-driven Proton Translocation by Primary Pumps

Abstract

A variety of unicellular microorganisms contain primary proton pumps that convert solar energy into a transmembrane electrochemical proton gradient, which is subsequently used by membrane ATP synthases to generate chemical energy. Well known examples of such pumps are the haloarchaeal rhodopsins, photoactive, seven-helix membrane proteins, which include the well studied proton pump bacteriorhodopsin (BR) 4 from Halobacterium salinarum and BR homologs in other haloarchaea. Recently, a much larger new family of light-driven proton pumps, the proteorhodopsins (PRs), was identified in marine proteobacteria throughout the oceans (1-3). Despite the diverse properties of PRs, including different visible absorption maxima and photocycle rates (4 -6), they all share with BR several key conserved residues as well as an all-trans-retinylidene chromophore in their unphotolyzed state, which is covalently bound to transmembrane helix G via a protonated Schiff base linkage.Many of the molecular events that occur in PRs following light activation are similar to those of BR, including an initial ultrafast all-trans313-cis-retinal isomerization, which triggers a sequence of protein conformational changes, including several intramolecular proton transfer reactions. The two key carboxylate groups involved in proton pumping in helix C of BR are conserved in PRs, and in the first found and most commonly studied PR, the Monterey Bay variant eBAC31A08, also known as green-absorbing proteorhodopsin (GPR), the helix C residues Asp-97 and Glu-108 undergo protonation changes during the photocycle similar to those of the homologous carboxylate residues in BR. Initial FTIR studies on GPR identified the role of Asp-97 as the Schiff base counterion and proton acceptor during Schiff base deprotonation and concomitant M formation and Glu-108 as the proton donor that reprotonates the Schiff base during N formation (7,8). Studies of other variants indicate these roles of the two carboxylic acid residues are general in the proteorhodopsin family. 5One major difference between BR and the PRs is the presence of a highly conserved histidine residue at position 75, near the middle of transmembrane helix B in the latter pigments. The His-75 homolog is not present in BR nor thus far found in other microbial rhodopsins (9). The proximity of His-75 to the pro-* This work was supported, in whole or in part, by National Institutes of Health Grants R01GM069969 (to K. J. R.) and R37GM27750 (to J. L. S.). This work was also supported by United States Department of Energy Grant DE-FG02-07ER15867 and The Robert A. Welch Foundation grant (to J. L. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Lipid Bilayer Composition Can Influence the Orientation of Proteorhodopsin in Artificial Membranes

“…However, densitometry calculations (not shown) reveal that total protein incorporation efficiency in a pH 5 environment is markedly less compared with incorporation at pH 6.2. Previous studies have shown that pR becomes unstable at lower pH values and causes precipitation (28). At pH 9, however, the C-terminal effective charge becomes À3.8 and the N-terminal remains unchanged at À2.…”

X-ray Crystallographic Structure of Thermophilic Rhodopsin

“…During the reaction, His 61 and Arg 92 are thought to be involved in the maintenance of both the pK a values of the charged residues and the proper structural changes of the peptide backbone analogous to the other eubacterial proton pumps including XR (18,(43)(44)(45)(46). Unfortunately, we cannot discuss the structural details of TR at the atomic level by precisely comparing them with XR or other microbial rhodopsins because of the present modest resolution (2.8 Å).…”

Assembling a Correctly Folded and Functional Heptahelical Membrane Protein by Protein Trans-splicing

“…Most importantly, the pK a of the primary proton acceptor Asp 97 is almost unaffected. Previous studies have shown that the protonation of Asp 97 in helix C is stabilized by an interaction with His 75 in helix B (23,35). In our case, helices B and C belong to two separately expressed fragments that are connected through the trans-splicing reaction.…”