Photoisomerization of the chromophore in bacteriorhodopsin during the proton pumping photocycle.
The configurations of the 13,14 double bond of the retinylidene chromophore in bacteriorhodopsin (bR) were investigated during the photocycle. The cycle was monitored by measuring the extent of formation of the M intermediate absorbing at 400-420 nm in the light and its rate of decay in the dark. It was inhibited by forming purple membrane films on glass and by using the synergistic ionophores beauvericin and valinomycin, in an equimolar ratio with bR, at 7 degrees C. These systems afforded enrichment in the M intermediate and a rate of decay that could be conveniently studied. The configuration was analyzed by extracting the chromophore with methylene chloride and measuring the ratio of 13-cis-to all-trans-retinals by high-performance liquid chromatography. The relationship between the percent bR cycling and percent 13-cis-retinal extracted was measured for the films by varying the light intensity; the molar ratio of bR cycling to 13-cis-retinal extracted was 1.11. This confirms previous studies which showed photoisomerization of the chromophore during the pumping cycle. The decay kinetics of the blue-shifted intermediate absorbing at 400-420 nm and of the 13-cis-retinal extracted were also measured for the purple membrane films and purple membrane suspensions containing the ionophores. The decay of the 13-cis-retinal extracted followed first-order kinetics with t 1/2 = 0.6 times the decay of the fastest measured component of the 400-420-nm decay. We conclude that the proton pumping photocycle of bR contains a photoisomerization about the 13,14 double bond of the chromophore from the all-trans to the 13-cis configuration and that the thermal rearrangement back to the all-trans form may occur more rapidly than the decay of the M photointermediate.[1]References
- Photoisomerization of the chromophore in bacteriorhodopsin during the proton pumping photocycle. Mowery, P.C., Stoeckenius, W. Biochemistry (1981) [Pubmed]
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