pH Dependence of the photocycle kinetics of the E46Q mutant of photoactive yellow protein: protonation equilibrium between I1 and I2 intermediates, chromophore deprotonation by hydroxyl uptake, and protonation relaxation of the dark state.
pH Dependence of the photocycle kinetics of the E46Q mutant of photoactive yellow protein: protonation equilibrium between I1 and I2 intermediates, chromophore deprotonation by hydroxyl uptake, and protonation relaxation of the dark state.
复制标题
光活性黄色蛋白 E46Q 突变体的光循环动力学的 pH 依赖性：I1 和 I2 中间体之间的质子化平衡、羟基摄取引起的发色团去质子化以及暗态的质子化弛豫。
DOI：
10.1021/bi034315d
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Heyn,MaartenP
中科院分区：
文献类型：
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作者：
Borucki,Berthold;Otto,Harald;Joshi,ChandraP;Gasperi,Chiara;Cusanovich,MichaelA;Devanathan,Savitha;Tollin,Gordon;Heyn,MaartenP
研究了PYP及其突变体E46Q和E46A的光循环动力学随pH的变化。E46是生色团的假定供体，生色团在I2中间体中质子化。对于E46Q，我们发现I2与其前体I1‘处于pH相关的平衡状态，pKaof为8.15，n=1。从这一结果和pH指示剂染料的实验中，我们得出结论，在I1’到I2的转变中，一个质子从外部介质中被吸收。PKaof8.15是在I1‘和I2之间的平衡状态下暴露在表面的发色团的pKaof8.15，与对羟基肉桂酸的酚基相近。与H+摄取相关的pH依赖的I1‘/I2平衡使人想起视紫红质信号状态形成过程中的MI/MII平衡。在此之上，pKano I2IS形成，并且I1‘以与pH无关的方式返回到初始状态P。在pH 4和pH 8之间，通过I2IS返回到P的衰减率与氢氧化物浓度(一阶)完全成正比，并且在这个转变中生色团的去质子化发生在氢氧化物吸收。在pKaof 8.15以上，由于I1‘的分支，返回P的表观速率常数是恒定的。与pH指示剂染料甲酚红在pH 8.3时的互补测量表明，剩余的仍通过I2循环的PYP分子在I2的形成中占据了一个质子。总之，这些观察结果提供了令人信服的证据，证明在光循环过程中，E46Q中的生色团从外部介质质子化和去质子化。对于黄色形式的突变体E46A，在[OH-]中返回P的表观速率常数在pH 8.3以下和pH 9.5以上也是线性的，I1‘的pKavue为8.8，这表明生色团质子化/去质子化的机制与E46Q相似。对于野生型，定性上也有相似的观察： 在碱性pH下I2的幅度降低，I1‘和I2处于平衡状态，I1’随着发色团的恢复而衰退，但阻止了pKaof I1‘的准确测定。我们估计它的值在11以上。基态P与低pH漂白形成的质子化发色团处于与pH有关的平衡中。对于E46Q和E46A，这些平衡的pKavue分别为4.8和7.9。当pH接近这些pKa时，光循环的动力学包含毫秒时间范围内的额外成分。利用pH跃迁停流实验表明，这些贡献是由于P的闪光激发引起P浓度的快速下降而引起的P/Pbl1平衡的驰豫，而P/Pbl1平衡的驰豫时间快于光周期时间是产生这种效应的条件。
The kinetics of the photocycle of PYP and its mutants E46Q and E46A were investigated as a function of pH. E46 is the putative donor of the chromophore which becomes protonated in the I2intermediate. For E46Q we find that I2is in a pH-dependent equilibrium with its precursor I1‘ with a pKaof 8.15 andn= 1. From this result and from experiments with pH indicator dyes, we conclude that in the I1‘ to I2transition one proton is taken up from the external medium. The pKaof 8.15 is that of the surface-exposed chromophore in the equilibrium between I1‘ and I2and is close to that of the phenolate group ofp-hydroxycinnamic acid. The pH-dependent I1‘/I2equilibrium with associated H+uptake is reminiscent of the MI/MIIequilibrium in the formation of the signaling state of rhodopsin. Well above this pKano I2is formed and I1‘ returns in a pH-independent manner to the initial state P. The decay rate for the return to P via I2is between pH 4 and pH 8, exactly proportional to the hydroxide concentration (first order), and the deprotonation of the chromophore in this transition occurs by hydroxide uptake. Well above the pKaof 8.15 the apparent rate constant for the return to P is constant due to the branching from I1‘. Complementary measurements with the pH indicator dye cresol red at pH 8.3 show that the remaining PYP molecules that still cycle via I2take up one proton in the formation of I2. Together, these observations provide compelling evidence that during the photocycle the chromophore in E46Q is protonated and deprotonated from the external medium. For the yellow form of the mutant E46A the apparent rate constant for the return to P is also linear in [OH-] below about pH 8.3 and constant above about pH 9.5, with a pKavalue of 8.8 for I1‘, suggesting a similar mechanism of chromophore protonation/deprotonation as in E46Q. For wild type qualitatively similar observations were made: the amplitude of I2decreased at alkaline pH, I1‘ and I2were in equilibrium, and I1‘ decayed together with the return to P. Chromophore hydrolysis prevented, however, an accurate determination of the pKaof I1‘. We estimate that its value is above 11. The ground state P is in the dark in a pH-dependent equilibrium with a low-pH bleached form Pblwith protonated chromophore. The pKavalues for these equilibria are 4.8 and 7.9 for E46Q and E46A, respectively. When the pH is close to these pKa's, the kinetics of the photocycle contains additional components in the millisecond time range. Using pH-jump stopped-flow experiments, we show that these contributions are due to the relaxation of the P/Pblequilibrium which is perturbed by the rapid decrease in the P concentration caused by the flash excitation of P. The condition for the occurrence of this effect is that the relaxation time of the P/Pblequilibrium is faster than the photocycle time.