pH dependence of the light-driven proton pumping by an archaerhodopsin from Tibet: comparison with bacteriorhodopsin
Ming Ming 1, Miao Lu 2, Sergei P Balashov 3, Thomas G. Ebrey 2, Qingguo Li 1 and Jiandong Ding 1*
1 Fudan University
2 University of Washington
3 University of California
* To whom correspondence should be addressed. E-mail: jdding1{at}fudan.edu.cn.
Submitted on October 22, 2005
Revised on December 30, 2005
Accepted on 19 January 2006
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Abstract |
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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 the 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 pKa of the proton release complex (PRC) in the M intermediate to be around 8.4, much higher than 5.7 of wide-type (WT) BR. The pH dependence of the rate constant of M formation shows that the pKa of PRC in the initial state of AR4 is about 10.4, whereas it is 9.7 in BR. Thus in AR4, the chromophore photoisomerization and subsequent proton transport from the Schiff base to Asp85 is coupled to a decrease in the pKa 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 pKa's of the key amino acid residues in AR4 were elevated compared to those of BR.
Key Words:
archaerhodopsin, bacteriorhodopsin, pH dependence, photocycle, proton pump
