Department of Microbiology and Molecular Genetics, Oklahoma State University , 307 Life Sciences East, Stillwater, Oklahoma 74078, United States.
Biochemistry. 2013 Oct 1;52(39):6824-33. doi: 10.1021/bi400930g. Epub 2013 Sep 16.
The active site of water oxidation in Photosystem II (PSII) is a Mn4CaO5 cluster that is located in a cavity between the D1 and CP43 protein subunits by which it is coordinated. The remainder of this cavity is filled with water molecules, which serve as a source of substrate and participate in poorly understood hydrogen bond networks that may modulate the function of the Mn4CaO5 cluster. These water molecules interact with the first and second sphere amino acid ligands to the Mn4CaO5 cluster and some water interacts directly with the Mn4CaO5 cluster. Here, the results of mutations to the amino acids that line the walls of several predicted cavities in the immediate vicinity of the Mn4CaO5 cluster were examined in Synechocystis sp. PCC 6803. Of these, mutations of Val185 in the D1 subunit resulted in the most interesting functional alterations. The hydrophobic D1-Val185 occupies a location contacting water molecules that are positioned between the redox active tyrosine (YZ) and the putative proton gate residue, D1-Asp61, and at a position opposite the oxo bridge atom, O5, of the cluster. Mutations of the residue D1-Val185 were produced, with the intention that the substitute residue would extend into the water cavity that includes H2O molecules that interact with the Mn4CaO5 cluster, amino acid ligands of the Mn4CaO5 cluster, YZ and the chloride co-factor of PSII. Three of these mutants, D1-Val185Asn, D1-Val185Thr, and D1-Val185Phe, were able to accumulate significant levels of charge separating PSII and were characterized using polarographic and fluorescent techniques. Of the three substitutions, the phenylalanine substitution was the most severe with a complete inability to evolve oxygen, despite being able to accumulate PSII and to undergo stable charge separation. The threonine substitution had no apparent effect on oxygen evolution other than a 40% reduction in the steady state rate of O2 production compared to the case of wild-type Synechocystis , due to a reduced ability to accumulate PSII centers. The asparagine substitution produced the most complex phenotype with respect to O2 evolution. Although still able to evolve oxygen, D1-Val185Asn does so less efficiently than wild-type PSII, with a higher miss factor than that for the wild type. Most significantly, asparagine substitution dramatically retards the rate of O2 release and results in an extension of the kinetic lag phase prior to O2 release that is highly reminiscent of the effects of mutations produced at D1-Asp61. The observed effects of the D1-Val185Phe and D1-Val185Asn mutations may be due to alterations in the environment of nearby chloride co-factor of PSII and/or alterations in the hydrogen bond network, perhaps impeding the movement of water to a binding site on the metal cluster.
PSII 中水分子氧化的活性部位是一个 Mn4CaO5 簇，它位于 D1 和 CP43 蛋白亚基之间的一个腔中，由其协调。该腔的其余部分充满了水分子，水分子作为底物的来源，并参与了理解不深的氢键网络，这些氢键网络可能调节 Mn4CaO5 簇的功能。这些水分子与 Mn4CaO5 簇的第一和第二配体氨基酸相互作用，一些水分子直接与 Mn4CaO5 簇相互作用。在这里，研究了 Synechocystis sp. PCC 6803 中 Mn4CaO5 簇附近几个预测腔壁氨基酸的突变结果。在这些突变中，D1 亚基中的 Val185 突变导致最有趣的功能改变。疏水的 D1-Val185 占据了一个位置，与位于氧化还原活性酪氨酸 (YZ) 和假定质子门残基 D1-Asp61 之间的水分子以及与簇的氧桥原子 O5 相对的位置接触。产生了 D1-Val185 残基的突变，目的是替代残基将延伸到包括与 Mn4CaO5 簇相互作用的水分子、Mn4CaO5 簇的氨基酸配体、YZ 和 PSII 的氯辅因子的水腔中。这三个突变体，D1-Val185Asn、D1-Val185Thr 和 D1-Val185Phe，能够积累显著水平的分离 PSII，并使用极谱和荧光技术进行了表征。在这三种取代中，苯丙氨酸取代是最严重的，尽管能够积累 PSII 并进行稳定的电荷分离，但完全不能进化氧气。与野生型 Synechocystis 相比，苏氨酸取代对氧气进化没有明显影响，除了 PSII 中心积累能力降低导致 O2 产生的稳态速率降低 40% 外。天冬酰胺取代对氧气进化产生了最复杂的表型。尽管仍然能够进化氧气，但 D1-Val185Asn 的效率低于野生型 PSII，错过因子高于野生型。最重要的是，天冬酰胺取代极大地减缓了 O2 释放的速度，并导致 O2 释放前动力学滞后阶段的延长，这非常类似于 D1-Asp61 突变产生的影响。D1-Val185Phe 和 D1-Val185Asn 突变的观察到的影响可能是由于 PSII 附近氯辅因子的环境改变和/或氢键网络的改变，可能阻碍了水分子向金属簇结合位点的移动。
