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pH Dependence of Light-Driven Proton Pumping by an Archaerhodopsin from Tibet: Comparison with Bacteriorhodopsin

Ming Ming ^*, Miao Lu , Sergei P. Balashov , Thomas G. Ebrey , Qingguo Li  and Jiandong Ding ^*

^* Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Fudan University, Shanghai, China; Department of Biology, University of Washington, Seattle, Washington; Department of Physiology and Biophysics, University of California, Irvine, California; and Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai, China

Correspondence: Address reprint requests to Jiandong Ding, E-mail: jdding1{at}fudan.edu.cn.

The pH-dependence of photocycle of archaerhodopsin 4 (AR4) was examined, and the underlying proton pumping mechanism investigated. AR4 is a retinal-containing membrane protein isolated from a strain of halobacteria from a Tibetan salt lake. It acts as a light-driven proton pump like bacteriorhodopsin (BR). However, AR4 exhibits an "abnormal" feature—the time sequence of proton release and uptake is reversed at neutral pH. We show here that the temporal sequence of AR4 reversed to "normal"—proton release preceding proton uptake—when the pH is increased above 8.6. We estimated the pK_a of the proton release complex (PRC) in the M-intermediate to be 8.4, much higher than 5.7 of wide-type BR. The pH-dependence of the rate constant of M-formation shows that the pK_a of PRC in the initial state of AR4 is 10.4, whereas it is 9.7 in BR. Thus in AR4, the chromophore photoisomerization and subsequent proton transport from the Schiff base to Asp-85 is coupled to a decrease in the pK_a of PRC from 10.4 to 8.4, which is 2 pK units less than in BR (4 units). This weakened coupling accounts for the lack of early proton release at neutral pH and the reversed time sequence of proton release and uptake in AR4. Nevertheless the PRC in AR4 effectively facilitates deprotonation of primary proton acceptor and recovery of initial state at neutral pH. We found also that all pK_as of the key amino acid residues in AR4 were elevated compared to those of BR.