Photoinduced proton release in proteorhodopsin at low pH: the possibility of a decrease in the pK(a) of Asp227

Area of Science:

• Biochemistry
• Photochemistry
• Microbiology

Background:

• Proteorhodopsin (PR) is a light-driven proton pump in marine bacteria.
• Previous studies explored PR proton transfer at pH 5-10.
• Low pH proton transfer mechanisms in PR remain less understood.

Purpose of the Study:

• Investigate photoinduced proton transfer in proteorhodopsin at low pH (<4).
• Identify key amino acid residues involved in proton release at acidic conditions.
• Characterize the role of Asp227 and Asp97 in proton transfer dynamics.

Main Methods:

• Utilized time-resolving pH electrodes (ITO/SnO2).
• Employed site-directed mutagenesis of proteorhodopsin.
• Performed spectroscopic analysis to determine pKa values.

Main Results:

• Observed a distinct proton release followed by uptake at low pH, without M intermediate formation.
• Asp227, the secondary counterion, is crucial for proton release.
• pKa of Asp227 decreased significantly upon photoexcitation, suggesting proton release.

Conclusions:

• Asp227 plays a critical role in photoinduced proton release at low pH.
• Negative charge stabilization of photoproducts involves Asp227 deprotonation, Cl- binding, or Asp97 deprotonation.
• Photoinduced proton release via pKa decrease of the secondary counterion is a conserved mechanism in microbial rhodopsins.