Ishida Naoko, Sugiura Miwa, Rappaport Fabrice, Lai Thanh-Lan, Rutherford A William, Boussac Alain
Institut de Biologie et Technologie de Saclay, URA CNRS 2096, Commissariat à l'Energie Atomique Saclay, 91191 Gif-sur-Yvette, France.
J Biol Chem. 2008 May 9;283(19):13330-40. doi: 10.1074/jbc.M710583200. Epub 2008 Mar 10.
The active site for water oxidation in photosystem II goes through five sequential oxidation states (S(0) to S(4)) before O(2) is evolved. It consists of a Mn(4)Ca cluster close to a redox-active tyrosine residue (Tyr(Z)). Cl(-) is also required for enzyme activity. To study the role of Ca(2+) and Cl(-) in PSII, these ions were biosynthetically substituted by Sr(2+) and Br(-), respectively, in the thermophilic cyanobacterium Thermosynechococcus elongatus. Irrespective of the combination of the non-native ions used (Ca/Br, Sr/Cl, Sr/Br), the enzyme could be isolated in a state that was fully intact but kinetically limited. The electron transfer steps affected by the exchanges were identified and then investigated by using time-resolved UV-visible absorption spectroscopy, time-resolved O(2) polarography, and thermoluminescence spectroscopy. The effect of the Ca(2+)/Sr(2+) and Cl(-)/Br(-) exchanges was additive, and the magnitude of the effect varied in the following order: Ca/Cl < Ca/Br < Sr/Cl < Sr/Br. In all cases, the rate of O(2) release was similar to that of the S(3)Tyr(Z)(.) to S(0)Tyr(Z) transition, with the slowest kinetics (i.e. the Sr/Br enzyme) being approximately 6-7 slower than in the native Ca/Cl enzyme. This slowdown in the kinetics was reflected in a decrease in the free energy level of the S(3) state as manifest by thermoluminescence. These observations indicate that Cl(-) is involved in the water oxidation mechanism. The possibility that Cl(-) is close to the active site is discussed in terms of recent structural models.
在光系统II中，水氧化的活性位点在氧气释放之前会经历五个连续的氧化态（S(0)到S(4)）。它由一个靠近氧化还原活性酪氨酸残基（Tyr(Z)）的Mn(4)Ca簇组成。酶活性也需要Cl(-)。为了研究Ca(2+)和Cl(-)在光系统II中的作用，在嗜热蓝细菌嗜热栖热放线菌中，分别用Sr(2+)和Br(-)对这些离子进行了生物合成替代。无论使用的非天然离子组合如何（Ca/Br、Sr/Cl、Sr/Br），该酶都可以以完全完整但动力学受限的状态分离出来。确定了受离子交换影响的电子转移步骤，然后通过时间分辨紫外可见吸收光谱、时间分辨O(2)极谱法和热发光光谱进行研究。Ca(2+)/Sr(2+)和Cl(-)/Br(-)交换的影响是累加的，影响程度按以下顺序变化：Ca/Cl < Ca/Br < Sr/Cl < Sr/Br。在所有情况下，O(2)释放速率与S(3)Tyr(Z)(.)到S(0)Tyr(Z)的转变速率相似，最慢的动力学（即Sr/Br酶）比天然Ca/Cl酶慢约6 - 7倍。动力学的这种减慢反映在热发光所显示的S(3)态自由能水平的降低上。这些观察结果表明Cl(-)参与了水氧化机制。根据最近的结构模型讨论了Cl(-)靠近活性位点的可能性。
