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

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 (pKa 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.

Coordinating the Structural Rearrangements Associated with Unidirectional Proton Transfer in the Bacteriorhodopsin Photocycle Induced by Deprotonation of the Proton-Release Group: A Time-Resolved Difference FTIR Spectroscopic Study

“…The decrease in intensity of these features at pH 5 is correlated with deprotonation of the PRG in M (p K a ∼6) (14). This is consistent with the observation that these negative bands were also abolished in the M-minus-BR spectrum at pH 10 (30), where the PRG is in a deprotonated state in both BR and M (20). Comparison of the spectra at neutral and acidic pH is based on idea that the reactions start from the same initial BR state, because the PRG is protonated at both pH 7 and 5.…”

“…A broad band between 1130 and 1045 cm -1 in the M-minus-BR spectrum at pH 7 is absent at pH 5. This band, which is also absent from the spectrum at pH 10, has been attributed to the vibrations of the side chain of Lys216 coupled with the vibration of C 15 —H of the retinal (30). Also, the M-minus-BR spectrum of wild type at pH 4, obtained by subtracting 35% of the spectrum of d 0 from the spectrum of d 2 at pH 4 (not shown), is identical in shape in the 1650-900 cm -1 region to the M-minus-BR spectrum at pH 5 (red line).…”

“…The water vibration band at 3657 cm -1 in the M-minus-BR spectrum at pH 7 (Figure 4, dotted line), which persists in the N-minus-BR spectrum, was shown to be less intense at pH 10 (30). The calculated M-minus-BR spectrum at pH 5 (solid line, see also Figure 2) shows a recognizable negative band at 3645 cm -1 superimposed on the tilted baseline produced by subtraction of an intense feature in the spectrum of d 0 .…”

“…The unstructured broad feature at 1730-1685 cm -1 was suggested to be due to backbone carbonyl groups interacting with C 15 -H of the retinal (30) and with the ring of Tyr57 (discussed in 30 based on Ref. 59).…”

“…Hence, FTIR spectroscopy is a powerful tool to detect chemical changes related to deprotonation of the PRG and the process that increases the proton affinity of Asp85. Since the p K a of the PRG in BR is ∼9 (20), the PRG will have already largely deprotonated in BR at pH 10, as in M. In comparing FTIR spectra at pH 7 and pH 10 (30), the contribution from the deprotonation of the PRG in M appeared with larger negative amplitudes in the spectrum at pH 7 than pH 10 in the 1730-1685 cm -1 region and the 1130-1045 cm -1 region, in addition to the 2120-1790 cm -1 region.…”