Aspartate鈥揌istidine Interaction in the Retinal Schiff Base Counterion of the Light-Driven Proton Pump of Exiguobacterium sibiricum

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• 作者：S. P. Balashov ; L. E. Petrovskaya ; E. P. Lukashev ; E. S. Imasheva ; A. K. Dioumaev ; J. M. Wang ; S. V. Sychev ; D. A. Dolgikh ; A. B. Rubin ; M. P. Kirpichnikov ; J. K. Lanyi
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：July 24, 2012
• 年：2012
• 卷：51
• 期：29
• 页码：5748-5762
• 全文大小：607K
• 年卷期：v.51,no.29(July 24, 2012)
• ISSN：1520-4995

文摘

One of the distinctive features of eubacterial retinal-based proton pumps, proteorhodopsins, xanthorhodopsin, and others, is hydrogen bonding of the key aspartate residue, the counterion to the retinal Schiff base, to a histidine. We describe properties of the recently found eubacterium proton pump from Exiguobacterium sibiricum (named ESR) expressed in Escherichia coli, especially features that depend on Asp鈥揌is interaction, the protonation state of the key aspartate, Asp85, and its ability to accept a proton from the Schiff base during the photocycle. Proton pumping by liposomes and E. coli cells containing ESR occurs in a broad pH range above pH 4.5. Large light-induced pH changes indicate that ESR is a potent proton pump. Replacement of His57 with methionine or asparagine strongly affects the pH-dependent properties of ESR. In the H57M mutant, a dramatic decrease in the quantum yield of chromophore fluorescence emission and a 45 nm blue shift of the absorption maximum with an increase in the pH from 5 to 8 indicate deprotonation of the counterion with a pK_a of 6.3, which is also the pK_a at which the M intermediate is observed in the photocycle of the protein solubilized in detergent [dodecyl maltoside (DDM)]. This is in contrast with the case for the wild-type protein, for which the same experiments show that the major fraction of Asp85 is deprotonated at pH >3 and that it protonates only at low pH, with a pK_a of 2.3. The M intermediate in the wild-type photocycle accumulates only at high pH, with an apparent pK_a of 9, via deprotonation of a residue interacting with Asp85, presumably His57. In liposomes reconstituted with ESR, the pK_a values for M formation and spectral shifts are 2鈥? pH units lower than in DDM. The distinctively different pH dependencies of the protonation of Asp85 and the accumulation of the M intermediate in the wild-type protein versus the H57M mutant indicate that there is strong Asp鈥揌is interaction, which substantially lowers the pK_a of Asp85 by stabilizing its deprotonated state.