Department of Physiology and Biophysics, University of California, Irvine, CA 92697, USA.
Biochemistry. 2012 Jul 24;51(29):5748-62. doi: 10.1021/bi300409m. Epub 2012 Jul 10.
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-His 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 2-3 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-His interaction, which substantially lowers the pK(a) of Asp85 by stabilizing its deprotonated state.
真细菌视紫红质质子泵、菌紫质和其他质子泵的一个显著特征是，与视黄醛席夫碱形成内盐的关键天冬氨酸残基与组氨酸形成氢键。我们描述了最近在极端嗜热古细菌 Exiguobacterium sibiricum（命名为 ESR）中发现的真细菌质子泵的性质，该质子泵在大肠杆菌中表达，特别是依赖于 Asp-His 相互作用、关键天冬氨酸残基 Asp85 的质子化状态及其在光循环中接受席夫碱基质子的能力的特征。含有 ESR 的脂质体和大肠杆菌细胞的质子泵通过在 pH 值高于 4.5 的宽 pH 范围内发生。大的光诱导 pH 变化表明 ESR 是一种有效的质子泵。用蛋氨酸或天冬酰胺取代 His57 会强烈影响 ESR 的 pH 依赖性性质。在 H57M 突变体中，与 pH 从 5 增加到 8 时发色团荧光发射量子产率的显著降低和吸收最大值的 45nm 蓝移表明，与 pK(a)为 6.3 的反离子去质子化，这也是在蛋白溶解在去污剂[十二烷基麦芽糖（DDM）]中的光循环中观察到 M 中间体的 pK(a)。这与野生型蛋白的情况形成对比，对于野生型蛋白，相同的实验表明，主要部分的 Asp85 在 pH>3 时去质子化，并且仅在低 pH 时质子化，pK(a)为 2.3。野生型光循环中的 M 中间体仅在高 pH 下通过与 Asp85 相互作用的残基去质子化而积累，推测为 His57，积累量高，表观 pK(a)为 9。在与 ESR 重建的脂质体中，M 形成和光谱位移的 pK(a)值比 DDM 低 2-3 pH 单位。野生型蛋白中 Asp85 的质子化和 M 中间体的积累与 H57M 突变体的 pH 依赖性明显不同，表明存在强烈的 Asp-His 相互作用，通过稳定其去质子化状态，大大降低了 Asp85 的 pK(a)。
