Evidence for the Rate of the Final Step in the Bacteriorhodopsin Photocycle Being Controlled by the Proton Release Group: R134H Mutant

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• 作者：Miao Lu ; Sergei P. Balashov ; Thomas G. Ebrey ; Ning Chen ; Yumei Chen ; Donald R. Menick ; and Rosalie K. Crouch
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：March 7, 2000
• 年：2000
• 卷：39
• 期：9
• 页码：2325 - 2331
• 全文大小：153K
• 年卷期：v.39,no.9(March 7, 2000)
• ISSN：1520-4995

文摘

Light absorbed by bacteriorhodopsin (bR) leads to a proton being released at the extracellularsurface of the purple membrane. Structural studies as well as studies of mutants of bR indicate that severalgroups form a pathway for proton transfer from the Schiff base to the extracellular surface. These groupsinclude D85, R82, E204, E194, and water molecules. Other residues may be important in tuning theinitial state pK_a values of these groups and in mediating light-induced changes of the pK_a values. Apotentially important residue is R134: it is located close to E194 and might interact electrostatically toaffect the pK_a of E194 and light-induced proton release. In this study we investigated effects of thesubstitution of R134 with a histidine on light-induced proton release and on the photocycle transitionsassociated with proton transfer. By measuring the light-induced absorption changes versus pH, we foundthat the R134H mutation results in an increase in the pK_a of the proton release group in both the M (0.6pK unit) and O (0.7 pK unit) intermediate states. This indicates the importance of R134 in tuning the pK_aof the group that, at neutral and high pH, releases the proton upon M formation (fast proton release) andthat, at low pH, releases the proton simultaneously with O decay (slow proton release). The higher pK_aof the proton release group found in R134H correlates with the slowing of the rate of the O rarr.gif"> bR transitionat low pH and probably is the cause of this slowing. The pH dependence of the fraction of the O intermediateis altered in R134H compared to the WT but is similar to that in the E194D mutant: a very small amountof O is present at neutral pH, but the fraction of O increases greatly upon decreasing the pH. Theseresults provide further support for the hypothesis that the O rarr.gif"> bR transition is controlled by the rate ofdeprotonation of the proton release group. These data also provide further evidence for the importance ofthe R134-E194 interaction in modulating proton release from D85 after light has led to its being protonated.