Water Structural Changes in the L and M Photocycle Intermediates of Bacteriorhodopsin as Revealed by Time-Resolved Step-Scan Fourier Transform Infrared (FTIR) Spectroscopy

Abstract

In previous FTIR studies of the photocycle intermediates of bacteriorhodopsin at cryogenic temperatures, water molecules were observed in the L intermediate, in the region surrounded by protein residues between the Schiff base and Asp96. In the M intermediate, the water molecules had moved away towards the Phe219-Thr46 region, but in the N intermediate they were again observed near the Schiff base. In order to evaluate the relevance of this scheme at room temperature, time resolved FTIR difference spectra of bacteriorhodopsin, including the water O-H stretching vibration frequency regions, were recorded in the μsec and msec time ranges. Vibrational changes of weakly-H-bonded water molecules were observed in L, M, and N. In each of these intermediates, the depletion of a water O-H stretching vibration at 3645 cm −1 originating from the initial unphotolzyed bacteriorhodopsin was observed as a trough in the difference spectrum. This vibration is due to the dangling O-H group of a water molecule which interacts with Asp85, and its absence in each of these intermediates indicates that there is perturbation of this O-H group. The formation of M is accompanied by the appearance of water O-H stretching vibrations at 3670 and 3657 cm −1 . These bands are due to water molecules present in the region surrounded by Thr46, Asp96 and Phe219. Formation of L at 298 K is accompanied by the perturbations of Asp96 and the Schiff base, though in different ways from what is observed at 170 K. Changes in a broad water vibrational feature, centered around 3610 cm −1 , are kinetically correlated with the L-to-M transition. These results imply that even at room temperature, water molecules interact with Asp96 and the Schiff base in L, though with a less rigid structure than at cryogenic temperatures.Bacteriorhodopsin is a light-driven proton pump. It undergoes a photocycle initiated by the absorption of light by a retinal chromophore which is covalently linked to Lys216 forming a protonated Schiff base. The initial consequence of light absorption is the trans-to-cis isomerization of the C 13 =C 14 bond of the retinal. Proton pumping takes place as the protein undergoes conformational changes from the initial unphotolyzed state with an all-trans retinal chromophore (BR 1 ) through a sequence of intermediates, and back to the BR state. Spectroscopic and kinetic studies, including FTIR studies have distinguished a sequence of + This work was supported by an NIH grant HL 16101 (to RBG).

Simultaneous measurements of fast optical and proton current kinetics in the bacteriorhodopsin photocycle using an enhanced spectrophotometer

“…Table 2 shows the presence of 6 kinetically distinct species with taus near 0.008, 0.05, 0.15, 0.5, 1.9, and 4.9 ms. These are essentially the same components as obtained previously by Hendler et al [14,20] and Chizhov et al [21] using optical data, and by Rodig et al [22] and Morgan et al [23 ] using FTIR data, and by Müller et al using both optical and FTIR data [24]. It is also shown in Table 2 that the component seen optically with a tau near 1.9 ms is absent in the electrical data.…”

Electrogenic Proton-Pumping Capabilities of the M-Fast and M-Slow Photocycles of Bacteriorhodopsin

“…There was no evidence of any significant amount of dark-adapted photocycle, such as the peak for BR being blue-shifted or the presence of an intermediate with a decay time constant near 30 ms. The fitted kinetic constants we obtained are in agreement with those published in the literature by other laboratories (10)(11)(12)(13). Instead of the previously described spectrophotometric system (14), a new updated version of the instrument, as described separately, was used (J. Biochem.…”

Crystal Structures of Different Substates of Bacteriorhodopsin's M Intermediate at Various pH Levels

“…This implies that the L-state structure at room temperature is not identical with that at 170 K, as has been suggested by Fourier transform infrared studies of purple membrane. 38 Conversely, the strong negative/positive densities around Glu194 and Glu204 can not be explained fully by the pH-dependent shift of the equilibrium between L and M. It is probable that the paired structure of Glu194 and Glu204 is not completely broken when the M state is generated at pH 4.4. This possibility will be discussed later.…”