Quantitative kinetic model for photoassembly of the photosynthetic water oxidase from its inorganic constituents: requirements for manganese and calcium in the kinetically resolved steps,.
Quantitative kinetic model for photoassembly of the photosynthetic water oxidase from its inorganic constituents: requirements for manganese and calcium in the kinetically resolved steps,.
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光合水氧化酶从其无机成分光组装的定量动力学模型：动力学解析步骤中对锰和钙的要求。
DOI：
10.1021/bi970187f
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Dismukes,GC
中科院分区：
文献类型：
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作者：
Zaltsman,L;Ananyev,GM;Bruntrager,E;Dismukes,GC
光激活过程是指高等植物光系统II载子蛋白和无机辅助因子（Mn2+、Ca2+和Cl-）组装成功能性水氧化复合物（WOC）的过程，从早期的研究中我们知道这是一个两步动力学过程，需要两个光诱导过程，中间间隔一个较慢的暗期。然而，这些步骤在任何动力学实验中都没有直接解决，直到超灵敏极谱o2电极的发展和用于去除辅助因子的改进螯合剂的合成使得第一个预稳态中间体的直接动力学解析成为可能[Ananyev， g.m. & Dismukes, g.c. (1996a)Biochemistry 35,4102−4109]。在本研究中，直接测定了菠菜PSII膜在钙和锰浓度范围内的光激活前两个光步骤和暗步骤的依赖速率，这些浓度至少比使用商业o2电极的浓度低10倍。得到了以下结果：(1)一个Mn2+离子在高亲和力位点与Mn3+结合并被光氧化，形成第一个光诱导中间体IM1。im1的形成与位于Mn位点的结合Ca2+离子的解离有关。(2)该位点对Ca2+解离的抑制常数为1.5 mM。(3)当钙浓度为8 mM时，该高亲和位点的Mn2+解离常数为8 μM，与PSII给电子的高亲和位点一致。(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，这样第二个Mn2+离子才能发生稳定的光氧化，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)该效应位点Ca2+结合恢复o2进化的Michaelis常数（Km）为1.4 mM，与完整PSII中o2进化所需钙的测量值相同。(6)从im1生成im2的低量子产率随着暗期的持续时间线性增加，直至我们所能检测到的最长暗期（10 s）。因此，第二个光解步骤的速率限制源于钙诱导的第一个中间体IM1的缓慢暗重排，我们认为这是一种蛋白质构象变化，允许下一个Mn2+离子的稳定结合。我们进一步提出，组装mn4簇所需的单个Ca2+离子相当于在完整的o2进化中心的“守门人”位点起作用的Ca2+离子，在那里它在限制底物进入mn4簇中起作用[Sivaraja， M.等人（1989），生物化学28,9459−9464；曹建军，等。（1991）生物化学30 (4)[J]。提出并讨论了光活化的分子模型。
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 O2electrode 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 O2electrodes. 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 IM1is 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 μM 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 O2evolution 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 O2evolution in intact PSII. (6) The low quantum yield for the formation of IM2from IM1increases 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 Mn4cluster 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 Mn4cluster [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.