High-resolution kinetic studies of the reassembly of the tetra-manganese cluster of photosynthetic water oxidation: Proton equilibrium, cations, and electrostatics
High-resolution kinetic studies of the reassembly of the tetra-manganese cluster of photosynthetic water oxidation: Proton equilibrium, cations, and electrostatics
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DOI：
10.1021/bi960894t
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发表时间：
1996-11-19
期刊：
影响因子：
2.9
通讯作者：
Dismukes, GC
中科院分区：
文献类型：
--
作者：
Ananyev, GM;Dismukes, GC
脉冲光活化动力学，即PSII的水氧化络合物（WOC）在必需无机辅助因子存在下的光诱导重组，已经用两种改进方法进行了研究：一种新的高效螯合剂N，N，N‘，N’-四丙酸-1,3-双（氨基甲基）苯（TPDBA），用于完全提取{Mn-4}和Ca2+，以及一种超灵敏的极谱电池用于O-2检测[Ananyev， G. M., & Dismukes， G. C.（1996）生物化学35,4102-4109]。测量了初始半衰期t(1/2)(第一中间体形成的滞后时间IM（1)加上第二中间体形成的半衰期IM(2)的总和）和O-2演化恢复的稳态产率Y-ss（与活性中心的数量成正比）。得出了以下结论：(1)阳离子（Ca2+， Mg2+和Na+）减缓了光激活的速度，尽管Ca2+是活性所必需的。似乎涉及两种不同的机制：与前两个Mn2+特异性位点中的一个或两个结合，以及通过静电转向筛选apo-WOC上负责集中Mn2+离子的负电荷；(2)在足够低的Mn2+浓度（8 μ M）下，Y-ss的钙需求量的米切里斯常数为K-m = 1.4 mM。从数值上看，在保留4个Mn离子的贫钙PSII膜中，O-2进化的再激活K-m是相同的；(3)在没有Ca2+的情况下，在饱和Mn2+ (8mn /apo-WOC)和Cl- (35mm)存在的情况下，稳定的四锰团簇既不会在光照下组装，也不会在随后加入CaCl2的黑暗中组装。然而，在达到最大Y-ss所需的钙浓度低于最佳浓度的情况下，光中会出现稳定但不活跃的钙依赖性团簇组装；(4)在pH为6.75和5.4之间，与缓冲液平衡的质子使半衰期大大增加了3倍，这表明在限速步骤（第二个Mn2+的光氧化）之前形成的第一个光氧化中间体的一个或多个质子电离；(5)已知完整WOC的还原剂亲脂性膜溶性阴离子四苯基硼（TPB-）将半衰期延长2.5倍（小于或等于40 μ M），并在20 μ M浓度下刺激Y-ss 50%。这些结果表明，TPB-增加了载子WOC附近Mn2+的局部浓度（Y-ss增加），同时在较高浓度下降低了完整WOC的S-2和S-3态（t（1/2）增加）。阴离子和阳离子的影响表明，克服膜/蛋白PSII复合物的表面电位可能在{Mn-4}簇重组动力学中起重要作用；(6) O-2演化动力学中Y-4/Y-3的比值随着O-2演化活性恢复程度的增加和Mn2+数量的增加而显著增大，表明底物水和过量Mn2+之间为减少功能化{Mn-4}簇而进行竞争。该比值通常反映了脱靶概率（α）。在这些结果的基础上，我们扩展了光激活模型，以包括H+和Ca2+在前两个中间体形成中的拮抗作用。
The kinetics of pulsed-light photoactivation, the light-induced reassembly of the water-oxidizing complex (WOC) of PSII in the presence of essential inorganic cofactors, has been studied using two improvements: a new efficient chelator, N,N,N',N'-tetrapropionate-1,3-bis(aminomethyl)benzene (TPDBA), for complete extraction of {Mn-4} and Ca2+ and an ultrasensitive polarographic cell for O-2 detection [Ananyev, G. M., & Dismukes, G. C. (1996) Biochemistry 35, 4102-4109]. Measurements have been made of the initial half-time, t(1/2) (sum of the lag time for formation of the first intermediate, IM(1), plus the half-time for formation of the second intermediate, IM(2)), and the steady-state yield, Y-ss, for recovery of O-2 evolution (proportional to the number of active centers). The following conclusions have been reached: (1) cations (Ca2+, Mg2+, and Na+) slow the rate of photoactivation, even though Ca2+ is essential for activity. Two distinct mechanisms appear to be involved: binding to one or both of the first two Mn2+-specific sites and screening of negative charges on apo-WOC that are responsible for concentrating Mn2+ ions by electrostatic steering; (2) the Michaelis constant for the calcium requirement for Y-ss at sufficiently low Mn2+ concentrations (8 mu M) that competition at the calcium site does not occur is K-m = 1.4 mM. Numerically, K-m is the same for reactivation of O-2 evolution in Ca-depleted PSII membranes which retain four Mn ions; (3) in the absence of Ca2+ but in the presence of saturating amounts of Mn2+ (8 Mn/apo-WOC) and Cl- (35 mM) assembly of a stable tetra-Mn cluster occurs neither under illumination nor in the dark after subsequent addition of CaCl2. However, in the presence of suboptimal concentrations of calcium required for maximum Y-ss, calcium-dependent assembly of stable yet inactive clusters occures in the light; (4) protons in equilibrium with the buffer greatly increase the half-time 3-fold between pH 6.75 and 5.4, indicating ionization of one or more protons from the first photo-oxidized intermediate formed prior to the rate-limiting step (photo-oxidation of the second Mn2+); (5) the lipophilic membrane soluble anion tetraphenylboron (TPB-), a known reductant of intact WOC, increases the half-time 2.5-fold (less than or equal to 40 mu M) and paradoxically stimulates Y-ss by 50% at 20 mu M concentration. These results suggest the TPB- increases the local concentration of Mn2+ adjacent to apo-WOC (Y-ss increase), while also reducing the S-2 and S-3 states of the intact WOC at higher concentrations (t(1/2) increase). The effects of anions and cations indicates that overcoming the surface potential of the membrane/protein PSII complex may play an important role in the kinetics of reassembly of the {Mn-4} cluster; (6) the ratio Y-4/Y-3 in the kinetics of O-2 evolution from a series of single-turnover flashes, a ratio that typically reflects the probability of misses (alpha), grows noticeably larger with increasing extent of recovery of O-2 evolving activity and also with increase in the amount of Mn2+, indication competition between substrate water and excess Mn2+ for reduction of the functional {Mn-4} cluster.On the basis of these results, we extend the model for photoactivation to include the antagonistic effects of H+ and Ca2+ in the formation of the first two intermediates.