Zaltsman L, Ananyev G M, Bruntrager E, Dismukes G C
Hoyt Laboratory, Department of Chemistry, Princeton University, Princeton, New Jersey 08544, USA.
Biochemistry. 1997 Jul 22;36(29):8914-22. doi: 10.1021/bi970187f.
The process of photoactivation, the assembly of a functional water-oxidizing complex (WOC) from the apoproteins of photosystem II of higher plants and inorganic cofactors (Mn2+, Ca2+, and Cl-), was known from earlier works to be a two-step kinetic process, requiring two light-induced processes separated by a slower dark period. However, these steps had not been directly resolved in any kinetic experiment, until development of an ultrasensitive polarographic O2 electrode and synthesis of an improved chelator for cofactor removal allowed direct kinetic resolution of the first pre-steady state intermediate [Ananyev, G. M. & Dismukes, G. C. (1996a) Biochemistry 35, 4102-4109]. Herein, the dependence of the rates of each of the first two light steps and the dark step of photoactivation was directly determined in spinach PSII membranes over a range of calcium and manganese concentrations at least 10-fold lower than those possible using commercial O2 electrodes. The following results were obtained. (1) One Mn2+ ion binds and is photooxidized to Mn3+ at a high-affinity site, forming the first light-induced intermediate, IM1. Formation of IM1 is coupled to the dissociation of a bound Ca2+ ion either located in the Mn site or coupled to it. (2) The inhibition constant for Ca2+ dissociation from this site is equal to 1.5 mM. (3) The dissociation constant of Mn2+ at this high-affinity site is equal to 8 microM at the optimum calcium concentration for O2-evolving activity of 8 mM, in agreement with the high-affinity site for electron donation to PSII. (4) Prior to the next photolytic step, one Ca2+ ion must bind at its effector site so that stable photooxidation of a second Mn2+ ion can occur, forming the second light-induced intermediate, IM2. This dark process is the rate-determining step. (5) The Michaelis constant for recovery of O2 evolution by Ca2+ binding at this effector site (Km) is equal to 1.4 mM, a value that is the same as that measured for the calcium requirement for O2 evolution in intact PSII. (6) The low quantum yield for the formation of IM2 from IM1 increases linearly with the duration of the dark period up to the longest period we could examine (10 s). Accordingly, the rate limitation in the second photolytic step originates from a slow calcium-induced dark rearrangement of the first intermediate, IM1, which we propose to be a protein conformational change that allows stable binding of the next Mn2+ ion. We further propose that the single Ca2+ ion which is required for assembly of the Mn4 cluster is equivalent to the Ca2+ ion which functions at the "gatekeeper" site in intact O2-evolving centers, where it plays a role in limiting substrate access to the Mn4 cluster [Sivaraja, M., et al. (1989) Biochemistry 28, 9459-9464; Tso, J., et al., (1991) Biochemistry 30, 4734-4739]. A molecular model for photoactivation is proposed and discussed.
光激活过程，即从高等植物光系统II的脱辅基蛋白和无机辅因子（Mn2+、Ca2+和Cl-）组装功能性水氧化复合物（WOC）的过程，早期研究已知其为两步动力学过程，需要两个光诱导过程，中间间隔一个较慢的暗期。然而，在任何动力学实验中，这些步骤都未得到直接解析，直到超灵敏极谱O2电极的开发以及用于去除辅因子的改进螯合剂的合成，才使得能够直接对第一个预稳态中间体进行动力学解析[阿纳涅夫，G. M. & 迪斯穆克斯，G. C.（1996a）《生物化学》35，4102 - 4109]。在此，在菠菜光系统II膜中，直接测定了光激活的前两个光步骤和暗步骤中每个步骤的速率，所涉及的钙和锰浓度范围至少比使用商业O2电极时低10倍。得到了以下结果。（1）一个Mn2+离子在高亲和力位点结合并被光氧化为Mn3+，形成第一个光诱导中间体IM1。IM1的形成与位于Mn位点或与之耦合的结合Ca2+离子的解离相关联。（2）该位点Ca2+解离的抑制常数等于1.5 mM。（3）在8 mM的O2释放活性最佳钙浓度下，该高亲和力位点处Mn2+的解离常数等于8 μM，这与向光系统II供电子的高亲和力位点一致。（4）在下一个光解步骤之前，一个Ca2+离子必须在其效应位点结合，以便第二个Mn2+离子能够发生稳定的光氧化，形成第二个光诱导中间体IM2。这个暗过程是速率决定步骤。（5）Ca2+在该效应位点结合以恢复O2释放的米氏常数（Km）等于1.4 mM，该值与完整光系统II中O2释放所需钙的测量值相同。（6）从IM1形成IM2的低量子产率随暗期持续时间线性增加，直至我们能够检测的最长时间（10 s）。因此，第二个光解步骤中的速率限制源于第一个中间体IM1的钙诱导暗重排缓慢，我们认为这是一种蛋白质构象变化，允许下一个Mn2+离子稳定结合。我们进一步提出，Mn4簇组装所需的单个Ca2+离子等同于完整O2释放中心“守门人”位点处起作用的Ca2+离子，在该位点它在限制底物进入Mn4簇方面发挥作用[Sivaraja，M.等人（1989）《生物化学》28，9459 - 9464；Tso，J.等人（1991）《生物化学》30，4734 - 4739]。提出并讨论了光激活的分子模型。
