Evidence for a halide-binding site in halorhodopsin.

Area of Science:

• Biophysics
• Membrane Protein Research
• Halobacterial Physiology

Background:

• Halorhodopsin (P580) functions as a light-driven chloride pump in halobacterial membranes.
• Understanding the halide-binding site is crucial for elucidating its transport mechanism.

Purpose of the Study:

• To characterize the halide-binding site in halorhodopsin.
• To investigate the influence of chloride and bromide on the pigment's photocycle and transport.
• To examine the effect of the diuretic drug MK-473 on halorhodopsin function.

Main Methods:

• Flash-induced absorption spectroscopy was used to monitor photointermediates.
• Halide concentrations were varied to observe effects on the photocycle.
• Transport activity was measured under sustained illumination.
• The effects of MK-473 were studied using kinetic analysis.

Main Results:

• In the absence of halide, P660 is the principal photointermediate (half-life ~1.5 ms).
• Chloride and bromide addition decrease P660 production and prolong its half-life, favoring P520 formation.
• Transport activity correlates with P520 accumulation up to ~400 mM halide, then declines.
• MK-473 competitively inhibits transport and photocycle progression, suggesting displacement of halide ions.

Conclusions:

• Chloride and bromide ions bind to distinct sites on halorhodopsin, affecting photointermediates P580, P660, and P520.
• Halide binding is essential for optimal transport activity.
• MK-473 acts as a competitive inhibitor by interacting with the halide-binding site.