His-75 in proteorhodopsin, a novel component in light-driven proton translocation by primary pumps.
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.
Bergo
Vladislav B
VB
Molecular Biophysics Laboratory, Photonics Center and Department of Physics, Boston University, Boston, Massachusetts 02215; Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center, Houston, Texas 77030.
Sineshchekov
Oleg A
OA
Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center, Houston, Texas 77030.
Kralj
Joel M
JM
Molecular Biophysics Laboratory, Photonics Center and Department of Physics, Boston University, Boston, Massachusetts 02215.
Partha
Ranga
R
Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center, Houston, Texas 77030.
Spudich
Elena N
EN
Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center, Houston, Texas 77030.
Rothschild
Kenneth J
KJ
Molecular Biophysics Laboratory, Photonics Center and Department of Physics, Boston University, Boston, Massachusetts 02215. Electronic address: kjr@bu.edu.
Spudich
John L
JL
Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center, Houston, Texas 77030. Electronic address: John.L.Spudich@uth.tmc.edu.
D000595:Amino Acid Sequence; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011522:Protons; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics; D025604:Rhodopsins, Microbial; D017550:Spectroscopy, Fourier Transform Infrared
D011522:Protons; D025604:Rhodopsins, Microbial; C413548:proteorhodopsin; D006639:Histidine; D012243:Rhodopsin