I. Introduction

The retinal protein halorhodopsin from the archaeon Halobacterium salinarumuses the energy of "green" light (500-650 nm, max at 578 nm) to transport chloride ions into the cell against the membrane potential. It functions as a light-driven anion pump, transporting beside chloride also other halides and nitrate into the cell and exemplifies vectorial catalysis.

The uptake of potassium chloride by cells is necessary to maintain osmotic balance during cell growth. A light-driven anion pump saves a substantial amount of metabolic energy.

Halorhodopsin has been analyzed in great detail and its structure is known to 1.8Å resolution. Its properties have been compared in great detail to those of bacteriorhodopsin to unravel similarities and differences of these two light-driven ion pumps which transport ions of opposite charge in opposite direction.

The further description is based on the review articles Oesterhelt (1995) and Oesterhelt (1998).

II. Structure and function of halorhodopsin

Halorhodopsin - as all retinal proteins from Halobacterium - folds into a seven-transmembrane helix topology with short interconnecting loops. The helices (named A-G) are arranged in an arc-like structure and tightly surround a retinal molecule that is covalently bound via a Schiff base to a conserved lysine (Lys-242) on helix G. The cross-section of HR with residues important for chloride transfer and the probable path of the anion is shown in Fig.1. The 3D structure is accessible through the PDB database, entry 1E12 which contains a bound chloride ion.

Retinal separates a cytoplasmic from an extracellular half channel that is lined by amino acids involved in anion transport by HR (especially the pair Thr-203/(Arg-200) in the cytoplasmic and the pair (Thr-111)/Arg-108 in the extracellular half channel). The Schiff base between retinal and Lys-242 is located at the center of this channel and chloride is bound in its vicinity. In halorhodopsin, the Schiff base remains protonated throughout the catalytic cycle as thermoisomerization of retinal is only possible in this state. To allow vectorial anion transport, the accessibility of the Schiff base for chloride must be switched during the catalytic cycle.

III. The catalytic cycle of halorhodopsin

Absorption of a photon by halorhodopsin initiates a catalytic cycle that leads to transport of an anion into the cell. Several intermediates in the catalytic cycle have been identified by spectroscopic techniques (Fig.2). By various biophysical experiments and comparison to bacteriorhodopsin, the changes in each step of the cycle have been determined and have been related to transport function.

The cycle can be formally described in terms of the six steps of isomerization (I), ion transport (T), and accessibility change (switch S). Retinal first photo-isomerizes from an all-trans to a 13-cis configuration followed by movement (or rearrangement) of the chloride ion in the extracellular half-channel to the Schiff base. To allow vectoriality, accessibility of the Schiff base is switched from extracellular to intracellular. The chloride ion moves probably to Thr-203. Retinal reisomerizes thermally and the accessibility of the Schiff base switches back to extracellular to reestablish the initial state. These steps represent the minimal number of steps needed to account for vectorial catalysis in halorhodopsin.

IV. The catalytic cycle step by step

  • primary reaction: the photoisomerization of retinal from all-trans to 13-cis
    In a stereoselective process, all-trans retinal is photoisomerized to 13-cis retinal and results in HR600 in ca 5 ps.

  • first chloride translocation step: from HR600 to HR520-I
    The state HR520 is reached from HR600 within several microseconds. This transition involves a slight change of the chloride position in the extracellular half-channel.

  • first accessibility switch reaction from extracellular to cytoplasmic: HR520-EC to HR520-CP
    To allow vectorial proton transport, accessibility of the Schiff base must occur from different sides of the membrane. This accessibility switch occurs at the level of the HR520 intermediate: HR520-EC to HR520-CP.

  • second chloride transfer step: from HR520-CP to HR640
    The chloride ion moves from the Schiff base via Thr-203 to the cytoplasmic surface during transformation from the HR520-CP to the HR640 intermediate within milliseconds.

  • thermoisomeration of retinal from 13-cis to all-trans and the second accessibility switch reaction from cytoplasmic to extracellular: HR640 to HR
    The transition of the HR640 to the ground state HR580 is the thermal 13-cis to all-trans isomerization of retinal and switching the accessibility of the Schiff base back from intracellular to extracellular. This occurs within several milliseconds and results in reformation of the initial state.

V. Site-directed mutagenesis of halorhodopsin

An important tool for the analysis of halorhodopsin structure and function is the possibility to produce specifically modified proteins by site directed mutagenesis and homologous overexpression. By this method the role of individual amino acid residues for the transport mechanism can be investigated.

Several mutations influence the kinetics of the photocycle and of chloride transport. The direct involvement of the amino acid pairs Thr-111/Arg-108 and Thr-203/Arg-200 in chloride transfer was analyzed with such mutants (Rüdiger and Oesterhelt (1997)).

VI. More detailed analysis in cooperations with other groups

In cooperation with external scientists, a more detailed analysis of halorhodopsin has been made. The description of these details is beyond the scope of this web site.

Cooperation partners are:

  • E. Bamberg

  • K. Gerwert

  • J. Heberle

  • R. Henderson

  • D.J. Müller

  • W. Zinth


  • Review article
    • D. Oesterhelt:
      Structure and Function of Halorhodopsin.
      Israel J. Chem. 35, 475-494 (1995).
    • D. Oesterhelt:
      The Structure and Mechanism of the Family of Retinal Proteins from Halophilic Archaea
      Curr. Op. Struct. Biol. 8, 489-500 (1998).
    • D. Oesterhelt:
      Photosynthese der zweiten Art: Die halophilen Archaea.
      In: Photosynthese (D. Häder, ed.) Georg Thieme Verlag, Stuttgart (1999) pp. 146-161.
  • 3D structure
  • photocycle
    • U. Haupts, J. Tittor, E. Bamberg and D. Oesterhelt:
      General Concept for Ion Translocation by Halobacterial Retinal Proteins: The Isomerization/Switch/Transfer Model.
      Biochemistry 36, 2-7 (1997)
    • P. Hegemann, D. Oesterhelt, M. Steiner:
      The Photocycle of the Chloride Pump Halorhodopsin. I. Azide Catalyzed Deprotonation of the Chromophore is a Side Reaction of Photocycle Intermediates Inactivating the Pump.
      EMBO J. 4, 2347-2350 (1985).
  • site-directed mutagenesis
  • nanotechnology
  • further references and classical papers
    • S. Paula, J. Tittor, D. Oesterhelt:
      Roles of Cytoplasmic Arginine and Threonine in Chloride Transport by the Bacteriorhodopsin Mutant D85T.
      Biophys. J. 80, 2386-2395 (2001).
    • E. Bamberg, J. Tittor, D. Oesterhelt:
      Light-driven Proton Pumping by Halorhodopsin.
      Proc.Natl. Acad. Sci. USA 90, 639-643 (1993).
    • A. Blanck, D. Oesterhelt:
      The Halo-Opsin Gene. II. Sequence, Primary Structure of Halorhodopsin and Comparison with Bacteriorhodopsin.
      EMBO J. 6, 265-273 (1987).
    • D. Oesterhelt, P. Hegemann, P. Tavan, K. Schulten:
      Trans-cis Isomerization of Retinal and a Mechanism for Ion Translocation in Halorhodopsin.
      Eur. Biophys. J. 14, 123-129 (1986).
    • P. Hegemann, D. Oesterhelt, E. Bamberg:
      The Transport Activity of the Light-Driven Chloride Pump Halorhodopsin is Regulated by Green and Blue Light.
      Biochim. Biophys. Acta 819, 195-205 (1985).
    • E. Bamberg, P. Hegemann, D. Oesterhelt:
      Reconstitution of the Light-driven Electrogenic Ion Pump Halorhodopsin.
      Biochim. Biophys. Acta 773, 53-60 (1984).
    • M. Steiner, D. Oesterhelt:
      Isolation and Properties of the Native Chromoprotein Halorhodopsin.
      EMBO J. 2, 1379-1385 (1983).
    • G. Wagner, D. Oesterhelt, G. Krippahl, J.K. Lanyi:
      Bioenergetic Role of Halorhodopsin in Halobacterium halobium Cells.
      FEBS Lett. 131, 341-345 (1981).
Go to Editor View