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首页> 外文期刊>Biochemistry >pH Dependence of the Photocycle Kinetics of the E46Q Mutant of Photoactive Yellow Protein: Protonation Equilibrium between I(1) and I(2) Intermediates, Chromophore Deprotonation by Hydroxyl Uptake, and Protonation Relaxation of the Dark State.
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pH Dependence of the Photocycle Kinetics of the E46Q Mutant of Photoactive Yellow Protein: Protonation Equilibrium between I(1) and I(2) Intermediates, Chromophore Deprotonation by Hydroxyl Uptake, and Protonation Relaxation of the Dark State.

机译：pH值取决于光活性黄色蛋白E46Q突变体的光循环动力学的：I（1）和I（2）中间体之间的质子化平衡，发色团被羟基吸收而去质子化以及黑暗状态的质子化弛豫。

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摘要

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 I(2) intermediate. For E46Q we find that I(2) is in a pH-dependent equilibrium with its precursor I(1)' with a pK(a) of 8.15 and n = 1. From this result and from experiments with pH indicator dyes, we conclude that in the I(1)' to I(2) transition one proton is taken up from the external medium. The pK(a) of 8.15 is that of the surface-exposed chromophore in the equilibrium between I(1)' and I(2) and is close to that of the phenolate group of p-hydroxycinnamic acid. The pH-dependent I(1)'/I(2) equilibrium with associated H(+) uptake is reminiscent of the M(I)/M(II) equilibrium in the formation of the signaling state of rhodopsin. Well above this pK(a) no I(2) is formed and I(1)' returns in a pH-independent manner to the initial state P. The decay rate for the return to P via I(2) is 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 pK(a) of 8.15 the apparent rate constant for the return to P is constant due to the branching from I(1)'. Complementary measurements with the pH indicator dye cresol red at pH 8.3 show that the remaining PYP molecules that still cycle via I(2) take up one proton in the formation of I(2). 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 pK(a) value of 8.8 for I(1)', suggesting a similar mechanism of chromophore protonation/deprotonation as in E46Q. For wild type qualitatively similar observations were made: the amplitude of I(2) decreased at alkaline pH, I(1)' and I(2) were in equilibrium, and I(1)' decayed together with the return to P. Chromophore hydrolysis prevented, however, an accurate determination of the pK(a) of I(1)'. 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 P(bl) with protonated chromophore. The pK(a) values for these equilibria are 4.8 and 7.9 for E46Q and E46A, respectively. When the pH is close to these pK(a)'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/P(bl) equilibrium 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/P(bl) equilibrium is faster than the photocycle time.
机译：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是生色团的推定供体，该生色团在I（2）中间体中被质子化。对于E46Q，我们发现I（2）与其前体I（1）'处于pH依赖的平衡状态，其pK（a）为8.15，n =1。根据该结果以及通过使用pH指示剂染料进行的实验，我们得出结论在I（1）'到I（2）的过渡中，一个质子从外部介质中吸收。 8.15的pK（a）是在I（1）'和I（2）之间平衡的表面暴露的发色团的pK（a），与对羟基肉桂酸的酚盐基团的接近。 pH相关的I（1）/ I（2）平衡以及相关的H（+）摄取，使视紫红质的信号传导状态形成了M（I）/ M（II）平衡状态。远高于此pK（a）不会形成I（2），I（1）'的pH值独立于初始状态P。通过I（2）返回P的衰减速率在pH 4之间pH值8与氢氧化物浓度（一阶）成正比，并且在此过渡过程中生色团的去质子化是由于氢氧化物的吸收而发生的。由于从I（1）'分支，远高于8.15的pK（a），返回P的视在速率常数恒定。用pH指示剂甲酚红在pH 8.3进行的补充测量表明，仍通过I（2）循环的其余PYP分子在I（2）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，即在光循环过程中，E46Q中的生色团被质子化并从外部介质中去质子化。对于黄色形式的突变体E46A，返回P的表观速率常数在[OH（-）]中在低于pH 8.3左右也是线性的，而在高于pH 9.5左右则恒定，I（p（k））值为8.8。 1）'，表明发色团质子化/去质子化的机理与E46Q类似。对于野生型，也进行了类似的定性观察：碱性条件下I（2）的振幅降低，I（1）'和I（2）处于平衡状态，I（1）'衰减并返回到P.发色团然而，水解阻止了I（1）′的pK（a）的精确测定。我们估计其值大于11。基态P在黑暗中处于pH依赖性平衡状态，带有低pH漂白形式P（bl）和质子化生色团。对于E46Q和E46A，这些平衡的pK（a）值分别为4.8和7.9。当pH值接近这些pK（a）时，光循环的动力学将在毫秒时间内包含其他成分。使用pH跳跃停流实验，我们表明这些贡献是由于P / P（bl）平衡的松弛所致，而P / P（bl）平衡的松弛是由P的瞬时激发引起的P浓度快速下降所扰动的。这种影响的发生是P / P（bl）平衡的弛豫时间比光循环时间快。

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