Formation of the M_N (M_open) Intermediate in the Wild-Type Bacteriorhodopsin Photocycle Is Accompanied by an Absorption Spectrum Shift to Shorter Wavelength, Like That in the Mutant D96N Bacteriorhodopsin Photocycle

A. N. Radionov, V. A. Klyachko, and A. D. Kaulen^*

Department of Photobiochemistry, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (7-095) 939-3181; E-mail: kaulen@phtbio.genebee.msu.su

^* To whom correspondence should be addressed.

Received June 4, 1999

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Maximum of the M intermediate difference spectrum in the wild-type Halobacterium salinarium purple membrane is localized at 405-406 nm under conditions favoring accumulation of the M_N intermediate (6 M guanidine chloride, pH 9.6), whereas immediately after laser flash the maximum is localized at 412 nm. The maximum is also localized at 412 nm 0.1 msec after the flash in the absence of guanidine chloride at pH 11.3. Within several milliseconds the maximum is shifted to short-wavelength region by 5-6 nm. This shift is similar to that in the D96N mutant which accompanies the M_N (M_open) intermediate formation. The main two differences are: 1) the rate of the shift is slower in the wild-type bacteriorhodopsin, and is similar to the rate of the M to N intermediate transition (t_1/2 ~ 2 msec); 2) the shift in the wild-type bacteriorhodopsin is observed at alkaline pH values which are higher than pK of the Schiff base (~10.8 at 1 M NaCl) in the N intermediate with the deprotonated Asp-96. Thus, the M_N (M_open) intermediate with open water-permeable inward proton channel is observed only at high pH, when the Schiff base and Asp-96 are deprotonated. The data confirmed our earlier conclusion that the M intermediate observed at lower pH has the closed inward proton channel.

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KEY WORDS: bacteriorhodopsin, photocycle, proton transport, purple membrane,Halobacterium salinarium

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