His-75 in Proteorhodopsin, a Novel Component in Light-driven Proton
Translocation by Primary
Pumps |
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Authors: | Vladislav B Bergo Oleg A Sineshchekov Joel M Kralj Ranga Partha Elena N Spudich Kenneth J Rothschild and John L Spudich |
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Institution: | ‡Molecular Biophysics Laboratory, Photonics Center and Department of Physics, Boston University, Boston, Massachusetts 02215 and the §Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center, Houston, Texas 77030 |
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Abstract: | Proteorhodopsins (PRs), photoactive retinylidene membrane proteins
ubiquitous in marine eubacteria, exhibit light-driven proton transport
activity similar to that of the well studied bacteriorhodopsin from halophilic
archaea. However, unlike bacteriorhodopsin, PRs have a single highly conserved
histidine located near the photoactive site of the protein. Time-resolved
Fourier transform IR difference spectroscopy combined with visible absorption
spectroscopy, isotope labeling, and electrical measurements of light-induced
charge movements reveal participation of His-75 in the proton translocation
mechanism of PR. Substitution of His-75 with Ala or Glu perturbed the
structure of the photoactive site and resulted in significantly shifted
visible absorption spectra. In contrast, His-75 substitution with a positively
charged Arg did not shift the visible absorption spectrum of PR. The mutation
to Arg also blocks the light-induced proton transfer from the Schiff base to
its counterion Asp-97 during the photocycle and the acid-induced protonation
of Asp-97 in the dark state of the protein. Isotope labeling of histidine
revealed that His-75 undergoes deprotonation during the photocycle in the
proton-pumping (high pH) form of PR, a reaction further supported by results
from H75E. Finally, all His-75 mutations greatly affect charge movements
within the PR and shift its pH dependence to acidic values. A model of the
proteorhodopsin proton transport process is proposed as follows: (i) in the
dark state His-75 is positively charged (protonated) over a wide pH range and
interacts directly with the Schiff base counterion Asp-97; and (ii)
photoisomerization-induced transfer of the Schiff base proton to the Asp-97
counterion disrupts its interaction with His-75 and triggers a histidine
deprotonation.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-trans→13-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 protein active site and specifically to the Schiff base counterion Asp-97
inferred from the x-ray crystal structure of BR suggests its involvement in
spectral tuning of the visible absorption
(10) and potentially PR
photochemical reactions. Because the pKa of histidine in
solution is close to neutral pH
(11), its imidazole group
often plays a major role in intramolecular proton transfers in enzymes,
including NADPH oxidase (12),
alcohol dehydrogenase (13),
carbonic anhydrase II (14),
and serine proteases (15).In this study we have used a combination of time-resolved FTIR difference
spectroscopy, visible absorption spectroscopy, isotope labeling, kinetic
charge displacement measurements, and site-directed mutagenesis to study the
role of His-75 in GPR. We report evidence that protonated His-75 interacts
directly with Asp-97 in the unphotolyzed protein and during the photocycle
undergoes a deprotonation in response to the protonation of Asp-97. |
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