Grove G N, Brudvig G W
Department of Chemistry, Yale University, New Haven, Connecticut 06520-8107, USA.
Biochemistry. 1998 Feb 10;37(6):1532-9. doi: 10.1021/bi971356z.
The identification of Ca2+ as a cofactor in photosynthetic O2 evolution has encouraged research into the role of Ca2+ in photosystem II (PSII). Previous methods used to identify the number of binding sites and their affinities were not able to measure Ca2+ binding at thermodynamic equilibrium. We introduce the use of a Ca2(+)-selective electrode to study equilibrium binding of Ca2+ to PSII. The number and affinities of binding sites were determined via Scatchard analysis on a series of PSII membrane preparations progressively depleted of the extrinsic polypeptides and Mn. Untreated PSII membranes bound approximately 4 Ca2+ per PSII with high affinity (K = 1.8 microM) and a larger number of Ca2+ with lower affinity. The high-affinity sites are assigned to divalent cation-binding sites on the light-harvesting complex II that are involved in membrane stacking, and the lower-affinity sites are attributed to nonspecific surface-binding sites. These sites were also observed in all of the extrinsic polypeptide- and Mn-depleted preparations. Depletion of the extrinsic polypeptides and/or Mn exposed additional very high-affinity Ca2(+)-binding sites which were not in equilibrium with free Ca2+ in untreated PSII, owing to the diffusion barrier created by the extrinsic polypeptides. Ca2(+)-depleted PSII membranes lacking the 23 and 17 kDa extrinsic proteins bound an additional 2.5 Ca2+ per PSII with K = 0.15 microM. This number of very high-affinity Ca2(+)-binding sites agrees with the previous work of Cheniae and co-workers [Kalosaka, K., et al. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., Ed.) pp 721-724, Kluwer, Dordrecht, The Netherlands] whose procedure for Ca2+ depletion was used. Further depletion of the 33 kDa extrinsic protein yielded a sample that bound only 0.7 very high-affinity Ca2+ per PSII with K = 0.19 microM. The loss of 2 very high-affinity Ca2(+)-binding sites upon depletion of the 33 kDa extrinsic protein could be due to a structural change of the O2-evolving complex which lost 2-3 of the 4 Mn ions in this sample. Finally, PSII membranes depleted of Mn and the 33, 23, and 17 kDa extrinsic proteins bound approximately 4 very high-affinity Ca2+ per PSII with K = 0.08 microM. These sites are assigned to Ca2+ binding to the vacant Mn sites.
钙离子作为光合放氧过程中的一种辅助因子被发现后，激发了人们对其在光系统II（PSII）中作用的研究。以往用于确定结合位点数量及其亲和力的方法无法在热力学平衡状态下测量钙离子的结合情况。我们引入了一种钙离子选择性电极来研究钙离子与PSII的平衡结合。通过对一系列逐步去除外在多肽和锰的PSII膜制剂进行Scatchard分析，确定了结合位点的数量和亲和力。未处理的PSII膜每PSII大约结合4个钙离子，其中高亲和力位点（K = 1.8 microM），还有大量低亲和力的钙离子结合位点。高亲和力位点被认为是位于捕光复合物II上参与膜堆叠的二价阳离子结合位点，低亲和力位点则归因于非特异性表面结合位点。在所有去除外在多肽和锰的制剂中也观察到了这些位点。去除外在多肽和/或锰后，暴露出了额外的超高亲和力钙离子结合位点，由于外在多肽形成的扩散屏障，这些位点在未处理的PSII中与游离钙离子处于非平衡状态。缺乏23 kDa和17 kDa外在蛋白的钙离子耗尽的PSII膜，每PSII额外结合2.5个钙离子，K = 0.15 microM。这种超高亲和力钙离子结合位点的数量与Cheniae及其同事之前的研究结果一致[Kalosaka, K., et al. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., Ed.) pp 721 - 724, Kluwer, Dordrecht, The Netherlands]，他们采用了相同的钙离子耗尽方法。进一步去除33 kDa外在蛋白后，得到的样品每PSII仅结合0.7个超高亲和力钙离子，K = 0.19 microM。去除33 kDa外在蛋白后，失去2个超高亲和力钙离子结合位点，可能是由于放氧复合物的结构变化，该样品中4个锰离子失去了2 - 3个。最后，去除锰以及33 kDa、23 kDa和17 kDa外在蛋白的PSII膜，每PSII大约结合4个超高亲和力钙离子，K = 0.08 microM。这些位点被认为是钙离子与空的锰位点结合。
