pH dependence of light-driven proton pumping by an archaerhodopsin from Tibet: comparison with bacteriorhodopsin.
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 approximately 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 approximately 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(a)s of the key amino acid residues in AR4 were elevated compared to those of BR.
Ming
Ming
M
Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Fudan University, Shanghai, China.
Lu
Miao
M
Balashov
Sergei P
SP
Ebrey
Thomas G
TG
Li
Qingguo
Q
Ding
Jiandong
J
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism; D006217:Halobacterium / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008027:Light; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017494:Proton Pumps / Q000737:chemistry* / Q000378:metabolism; D013053:Spectrophotometry; D018609:Tibet
D019843:Archaeal Proteins; D017494:Proton Pumps; C055476:archaerhodopsin protein, Archaea; D001436:Bacteriorhodopsins