Kuntzleman Thomas, Yocum Charles F
Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109-1048, USA.
Biochemistry. 2005 Feb 15;44(6):2129-42. doi: 10.1021/bi048460i.
Hydroxylamine and hydroquinone were used to probe the oxidation states of Mn in the oxygen-evolving complex of dark-adapted intact (hydroxylamine) and salt-washed (hydroquinone) photosystem II. These preparations were incubated in the dark for 24 h in the presence of increasing reductant/photosystem II ratios, and the loss of oxygen evolution activity and of Mn(II) was determined for each incubation mixture. Monte Carlo simulations of these data yielded models that provide insight into the structure, reactivity, and oxidation states of the manganese in the oxygen-evolving complex. Specifically, the data support oxidation states of Mn(III)(2)/Mn(IV)(2) for the dark stable S(1) state of the O(2)-evolving complex. Activity and Mn(II) loss data were best modeled by assuming an S(1) --> S(-)(1) conversion of intermediate probability, a S(-)(1) --> S(-)(3) reaction of high probability, and subsequent step(s) of low probability. This model predicts that photosystem II Mn clusters that have undergone an initial reduction step become more reactive toward a second reduction, followed by a slower third reduction step. Analysis of the Mn(II) release parameters used to model the data suggests that the photosystem II manganese cluster consists of three Mn atoms that exhibit a facile reactivity with both reductants, and a single Mn that is reducible but sterically trapped at or near its binding site. Activity assays indicate that intact photosystem II centers reduced to S(-)(1) can evolve oxygen upon illumination, but that these centers are inactive in preparations depleted of the extrinsic 23 and 17 kDa polypeptides. Finally, it was found that a substantial population of the tyrosine D radical is reduced by hydroxylamine, but a smaller population reacts with hydroquinone over the course of a 24 h exposure to the reductant.
用羟胺和对苯二酚来探测黑暗适应的完整（羟胺）和盐洗（对苯二酚）光系统II放氧复合体中锰的氧化态。将这些制剂在黑暗中于不断增加的还原剂/光系统II比例存在下孵育24小时，并测定每个孵育混合物中放氧活性和锰（II）的损失。对这些数据的蒙特卡洛模拟产生了一些模型，这些模型有助于深入了解放氧复合体中锰的结构、反应性和氧化态。具体而言，数据支持放氧复合体黑暗稳定S(1)态的Mn(III)(2)/Mn(IV)(2)氧化态。活性和锰（II）损失数据通过假设中间概率的S(1) --> S(-)(1)转化、高概率的S(-)(1) --> S(-)(3)反应以及随后低概率的步骤来进行最佳建模。该模型预测，经历了初始还原步骤的光系统II锰簇对第二次还原变得更具反应性，随后是较慢的第三次还原步骤。对用于模拟数据的锰（II）释放参数的分析表明，光系统II锰簇由三个与两种还原剂都表现出容易反应性的锰原子和一个可还原但在其结合位点或附近空间受阻的单个锰原子组成。活性测定表明，还原到S(-)(1)的完整光系统II中心在光照下可以放氧，但这些中心在去除了外在的23 kDa和17 kDa多肽的制剂中无活性。最后，发现相当一部分酪氨酸D自由基被羟胺还原，但在24小时暴露于还原剂的过程中，与对苯二酚反应的比例较小。
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