Jin Lu, Smith Paul, Noble Christopher J, Stranger Rob, Hanson Graeme R, Pace Ron J
Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.
Phys Chem Chem Phys. 2014 May 7;16(17):7799-812. doi: 10.1039/c3cp55189j.
We report the first (55)Mn pulsed ENDOR studies on the S2 state multiline spin ½ centre of the oxygen evolving complex (OEC) in Photosystem II (PS II), at temperatures below 4.2 K. These were performed on highly active samples of spinach PS II core complexes, developed previously in our laboratories for photosystem spectroscopic use, at temperatures down to 2.5 K. Under these conditions, relaxation effects which have previously hindered observation of most of the manganese ENDOR resonances from the OEC coupled Mn cluster are suppressed. (55)Mn ENDOR hyperfine couplings ranging from ∼50 to ∼680 MHz are now seen on the S2 state multiline EPR signal. These, together with complementary high resolution X-band CW EPR measurements and detailed simulations, reveal that at least two and probably three Mn hyperfine couplings with large anisotropy are seen, indicating that three Mn(III) ions are likely present in the functional S2 state of the enzyme. This suggests a low oxidation state paradigm for the OEC (mean Mn oxidation level 3.0 in the S1 state) and unexpected Mn exchange coupling in the S2 state, with two Mn ions nearly magnetically silent. Our results rationalize a number of previous ligand ESEEM/ENDOR studies and labelled water exchange experiments on the S2 state of the photosystem, in a common picture which is closely consistent with recent photo-assembly (Kolling et al., Biophys. J. 2012, 103, 313-322) and large scale computational studies on the OEC (Gatt et al., Angew. Chem., Int. Ed. 2012, 51, 12025-12028, Kurashige et al. Nat. Chem. 2013, 5, 660-666).
我们报道了在温度低于4.2K时，对光系统II（PS II）中析氧复合物（OEC）的S2态多线自旋1/2中心进行的首次（55）Mn脉冲ENDOR研究。这些研究是在我们实验室先前为光系统光谱学用途开发的高活性菠菜PS II核心复合物样品上进行的，温度低至2.5K。在这些条件下，先前阻碍观察来自OEC耦合锰簇的大多数锰ENDOR共振的弛豫效应得到抑制。现在在S2态多线EPR信号上可以看到范围从约50到约680MHz的（55）Mn ENDOR超精细耦合。这些结果与互补的高分辨率X波段连续波EPR测量和详细模拟一起表明，至少可以看到两个且可能三个具有大各向异性的锰超精细耦合，这表明在该酶的功能性S2态中可能存在三个Mn(III)离子。这表明OEC具有低氧化态模式（S1态中平均锰氧化水平为3.0），并且在S2态中存在意想不到的锰交换耦合，其中两个锰离子几乎没有磁性。我们的结果在一个与最近的光组装（Kolling等人，《生物物理杂志》，2012年，103卷，313 - 322页）和关于OEC的大规模计算研究（Gatt等人，《德国应用化学》，国际版，2012年，51卷，12025 - 12028页，Kurashige等人，《自然化学》，2013年，5卷，660 - 666页）密切一致的共同图景中，使先前一些关于光系统S2态的配体ESEEM/ENDOR研究和标记水交换实验合理化。
