Fourier transform infrared double-flash experiments resolve bacteriorhodopsin's M1 to M2 transition

Area of Science:

• Biophysics
• Structural Biology
• Spectroscopy

Background:

• Bacteriorhodopsin (bR) functions as a light-driven proton pump, essential for cellular energy generation.
• The directional proton transfer relies on the precise orientation of the protonated Schiff base (PSB).
• The M intermediate's M1 to M2 transition is hypothesized to involve a 'reprotonation switch' critical for PSB orientation.

Purpose of the Study:

• To elucidate the molecular mechanisms underlying the M1 to M2 transition in bacteriorhodopsin.
• To investigate the structural changes and proton accessibility configurations of the PSB during this transition.
• To resolve the dynamics of Schiff base reprotonation in the M photo-back-reaction.

Main Methods:

• Utilized double-flash experiments to selectively probe M1 and M2 intermediates.
• Employed nanosecond step-scan Fourier transform infrared (FTIR) spectroscopy to monitor spectral changes.
• Analyzed FTIR difference spectra (BR-M1 and BR-M2) to identify structural and protonation state changes.

Main Results:

• Observed only minor backbone movements (1-2 peptide bonds) during the M1 to M2 transition, distinct from the M to N transition.
• Identified altered interactions between the Schiff base and Y185 in the M2 intermediate.
• Found no evidence of chromophore isomerization during the M1 to M2 transition.
• Demonstrated time-resolved Schiff base reprotonation from D85 in the M photo-back-reaction, differing from the canonical D96 pathway.

Conclusions:

• The M1 to M2 transition involves subtle backbone rearrangements that reconfigure the PSB's proton accessibility.
• These findings clarify the reprotonation switch mechanism and its role in proton pump directionality.
• The observed alternative reprotonation pathway from D85 challenges the established model for the M photo-back-reaction.