Structural Changes in Bacteriorhodopsin following Retinal Photoisomerization from the 13-Cis Form

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Abstract
Bacteriorhodopsin (BR), a light-driven proton pump in Halobacterium salinarum, accommodates two resting forms of the retinylidene chromophore, the all-trans form (AT-BR) and the 13-cis,15-syn form (13C-BR). Both isomers are present in thermal equilibrium in the dark, but only the all-trans form has proton-pump activity. In this study, we applied low-temperature Fourier-transform infrared (FTIR) spectroscopy to 13C-BR at 77 K and compared the local structure around the chromophore before and after photoisomerization with that in AT-BR. Strong hydrogen-out-of-plane (HOOP) vibrations were observed at 964 and 958 cm-1 for the K state of 13C-BR (13C-BRK) versus a vibration at 957 cm-1 for the K state of AT-BR (AT-BRK). In AT-BRK, but not in 13C-BRK, the HOOP modes exhibit isotope shifts upon deuteration of the retinylidene at C15 and at the Schiff base nitrogen. Whereas the HOOP modes of AT-BRK were significantly affected by the mutation of Thr89, this was not the case for the HOOP modes of 13C-BRK. These observations imply that, while the chromophore distortion is localized near the Schiff base in AT-BRK, it is located elsewhere in 13C-BRK. By use of [ζ-15N]lysine-labeled BR, we identified the N−D stretching vibrations of the 13C-BR Schiff base (in D2O) at 2173 and 2056 cm-1, close in frequency to those of AT-BR. These frequencies indicate strong hydrogen bonding of the Schiff base in 13C-BR, presumably with a water molecule as in AT-BR. In contrast, the N−D stretching vibration appears at 2332 and 2276 cm-1 in 13C-BRK versus values of 2495 and 2468 cm-1 for AT-BRK, suggesting that the rupture of the Schiff base hydrogen bond that occurs in AT-BRK does not occur in 13C-BRK. Rotational motion of the Schiff base upon retinal isomerization is probably smaller in magnitude for 13C-BR than for AT-BR. These differences in the primary step are possibly related to the absence of light-driven proton pumping by 13C-BR.