Local-access model for proton transfer in bacteriorhodopsin

Area of Science:

• Biophysics
• Molecular Biology
• Protein Chemistry

Background:

• Bacteriorhodopsin is a light-driven proton pump crucial for cellular energy.
• The Schiff base is a key intermediate in the proton transfer pathway.
• Understanding proton transfer mechanisms in mutants lacking proton acceptors/donors is vital.

Purpose of the Study:

• To investigate the accessibility of the retinal Schiff base in the D85N/D96N bacteriorhodopsin mutant.
• To elucidate the proton transfer switch mechanism in the absence of key proton donor/acceptor residues.
• To differentiate between extracellular (EC) and cytoplasmic (CP) access routes for proton transfer.

Main Methods:

• Studied protonation/deprotonation of the Schiff base using pH jump and photocycle experiments.
• Measured proton transfer rates in visible and infrared spectroscopy.
• Utilized millimolar azide concentrations to determine EC vs. CP access.

Main Results:

• In metastable photoproducts, Schiff base access flickers between EC and CP directions.
• In stable isomeric states of the D85N/D96N mutant, Schiff base access is locked to specific directions (EC or CP).
• Reisomerization of retinal, not half-channel conductivity, appears to function as the proton transfer switch in stable states.

Conclusions:

• A new hypothesis for the proton-transfer switch involves flickering access in metastable states and locked access in stable states.
• Proton transfer direction is determined by local access, donor/acceptor groups (wild-type), or half-channel conductivity (mutant).
• Retinal reisomerization plays a critical role in the proton transfer switch mechanism in the D85N/D96N mutant.