Proton binding within a membrane protein by a protonated water cluster

Area of Science:

• Biochemistry
• Structural Biology
• Spectroscopy

Background:

• Proton transfer is fundamental to numerous enzymatic processes.
• Bacteriorhodopsin (bR) utilizes a photocycle for proton pumping.
• Water molecules are increasingly recognized for their roles in protein function.

Purpose of the Study:

• To investigate the role of water networks as proton-binding sites in proteins.
• To characterize the proton release site in bacteriorhodopsin using spectroscopic methods.
• To elucidate the influence of specific amino acid residues on the protonated water network and proton release mechanism.

Main Methods:

• Fourier transform infrared (FTIR) spectroscopy, including in situ hydrogen/deuterium exchange.
• Site-directed mutagenesis of bacteriorhodopsin.
• Analysis of IR continuum absorbance changes during the bR photocycle.

Main Results:

• A protonated water cluster, comprising one proton and approximately five water molecules, was identified at the bR proton release site.
• This water cluster is stabilized by six side chains and three backbone groups.
• Mutations affecting key residues perturbed proton release, highlighting their role in stabilizing the water network.
• In situ H/D exchange FTIR revealed a switch from a delocalized to a localized proton-binding site in mutated bR.

Conclusions:

• Water networks can serve as critical proton-binding sites in proteins, complementing traditional amino acid roles.
• The identified water cluster and surrounding residues are essential for efficient proton release in bacteriorhodopsin.
• Mutations can alter the proton binding and release mechanism, shifting from delocalized to localized protonation states.