Proton transport by halorhodopsin

Area of Science:

• Membrane protein biophysics
• Photobiology
• Ion transport mechanisms

Background:

• Halorhodopsin (NpHR) from Natronobacterium pharaonis functions as a light-driven chloride pump.
• The chloride binding site in NpHR also accommodates azide ions.
• Bacteriorhodopsin (BR) is a well-characterized light-driven proton pump.

Purpose of the Study:

• To investigate the functional consequences of azide binding to halorhodopsin.
• To explore the potential for halorhodopsin to exhibit proton pumping activity.
• To compare the photocycle of azide-bound halorhodopsin with that of bacteriorhodopsin.

Main Methods:

• Spectroscopic analysis using pyranine to detect proton release.
• Electrophysiological measurements using a potential-sensitive electrode.
• Biochemical assays measuring light-dependent pH changes in vesicle suspensions.

Main Results:

• Azide binding to halorhodopsin induces transient deprotonation of the retinal Schiff base upon photoexcitation.
• A bacteriorhodopsin-like photocycle is observed, including proton release and reprotonation kinetics dependent on azide.
• Active electrogenic proton transport from cytoplasmic to extracellular space is detected.
• Azide molecules fulfill roles analogous to Asp-85 and Asp-96 in bacteriorhodopsin.

Conclusions:

• Halorhodopsin possesses the structural capacity for proton pumping, modulated by azide binding.
• Azide binding transforms halorhodopsin into a proton pump, highlighting functional plasticity in these proteins.
• This study reveals shared structural elements and functional potential between chloride and proton pumps.