Structural changes due to the deprotonation of the proton release group in the M-photointermediate of bacteriorhodopsin as revealed by time-resolved FTIR spectroscopy

Joel E. Morgan, Ahmet S. Vakkasoglu, Johan Lugtenburg, Robert B. Gennis, Akio Maeda

Research output: Contribution to journalArticlepeer-review

Abstract

One of the steps in the proton pumping cycle of bacteriorhodopsin (BR) is the release of a proton from the proton-release group (PRG) on the extracellular side of the Schiff base. This proton release takes place shortly after deprotonation of the Schiff base (L-to-M transition) and results in an increase in the pKa of Asp85, which is a crucial mechanistic step for one-way proton transfer for the entire photocycle. Deprotonation of the PRG can also be brought about without photoactivation, by raising the pH of the enzyme (pK a of PRG; ∼9). Thus, comparison of the FTIR difference spectrum for formation of the M intermediate (M minus initial unphotolyzed BR state) at pH 7 to the corresponding spectrum generated at pH 10 may reveal structural changes specifically associated with deprotonation of the PRG. Vibrational bands of BR that change upon M formation are distributed across a broad region between 2120 and 1685 cm-1. This broad band is made up of two parts. The band above 1780 cm-1, which is insensitive to C 15-deuteration of the retinal, may be due to a proton delocalized in the PRG. The band between 1725 and 1685 cm-1, on the lower frequency side of the broad band, is sensitive to C15-deuteration. This band may arise from transition dipole coupling of the vibrations of backbone carbonyl groups in helix G with the side chain of Tyr57 and with the C15-H of the Schiff base. In M, these broad bands are abolished, and the 3657 cm -1 band, which is due to the disruption of the hydrogen bonding of a water molecule, probably with Arg82, appears. Loss of the interaction of the backbone carbonyl groups in helix G with Tyr57 and the Schiff base, and separation of Tyr57 from Arg82, may be causes of these spectral changes, leading to the stabilization of the protonated Asp85 in M.

Original languageEnglish (US)
Pages (from-to)11598-11605
Number of pages8
JournalBiochemistry
Volume47
Issue number44
DOIs
StatePublished - Nov 4 2008

ASJC Scopus subject areas

  • Biochemistry

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