Structural Changes Due to the Deprotonation of the Proton Release Group in the M-Photointermediate of Bacteriorhodopsin as Revealed by Time-Resolved FTIR Spectroscopy

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• 作者：Joel E. Morgan ; Ahmet S. Vakkasoglu ; Johan Lugtenburg ; Robert B. Gennis ; Akio Maeda
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：November 4, 2008
• 年：2008
• 卷：47
• 期：44
• 页码：11598-11605
• 全文大小：215K
• 年卷期：v.47,no.44(November 4, 2008)
• ISSN：1520-4995

文摘

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 pK_a 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 C₁₅-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.