Assembly of the tetra-Mn site of photosynthetic water oxidation by photoactivation: Mn stoichiometry and detection of a new intermediate
Assembly of the tetra-Mn site of photosynthetic water oxidation by photoactivation: Mn stoichiometry and detection of a new intermediate
复制标题
DOI：
10.1021/bi952667h
复制
发表时间：
1996-04-02
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
光活化的过程，即光系统II(PSII)膜的水氧化复合体(WOC)的组装，已经通过两个主要的方法学改进进行了研究。首先，使用了一种新的亲脂性螯合剂N，N，N‘，N’-tetrapropionato-1，3-bis(aminomethyl)benzene(TPDBA)，它允许完全提取锰和钙以及三种外源WOC多肽，同时将对apo-PSII蛋白的损害降至最低，而且重要的是，消除了使用还原剂的需要。其次，建立了超灵敏、快速响应的极谱电池和检测系统。该设备包括：(A)用于控制光强度、脉冲持续时间和暗间隔的超亮红色发光二极管(LED)，其特征是最大限度地减少光抑制；(B)微型(5亩L)O-2极谱池(克拉克型)，装有用于快速响应(100ms)的薄硅膜；以及(C)集成到微单元中并连接到带通交流放大器的DC/AC前置放大器。这种灵敏度可以在信噪比=5/1的情况下，在每个闪光中检测到接近5×10(-14)摩尔的O-2。这些改进使得可以探测到100倍的低锰浓度。在最佳条件下，与去除三种外源多肽的PSII膜(35%V-max vs完整PSII)相比，放氧活性可以完全恢复。光活化稳态O-2产率Y-ss和恢复半衰期t(1/2)与Mn浓度的滴定结果表明，在Y-ss的95%恢复时，4.0mN/P680被协同吸收，在稳态条件下，1.1-1.2mN原子参与了限速光解步骤。由于光抑制的最小化，这种中间体在整个PSII中心种群中表现出单一的指数恢复动力学。当锰原子超过4mN/P680时，光活化速率加快，但超过8-10mN/P680时，光活化效率降低。在相同的电化学势下，Y-ss和t(1/2)都出现极大值，分别为380 mV和340 mV。我们将这一最大值归因于D1多肽(Y-z(+))的氧化还原活性酪氨酸-161与电子受体(可能是细胞色素b(559))之间的复合反应的消除，或者是光活化中中间体的稳定。在较低的Mn2+浓度下，可以直接观察到一个新的稳定前的动力学中间体，它与少于4个的Mn原子结合。这一早期动力学相的速率依赖于锰的浓度，与电子受体的一致性和浓度无关。
The process of photoactivation, the assembly of the water-oxidizing complex (WOC) of photosystem II (PSII) membranes, has been examined using two major improvements in methodology. First, a new lipophilic chelator, N,N,N',N'-tetrapropionato-1,3-bis(aminomethyl)benzene (TPDBA), has been used that permits complete extraction of both manganese and calcium and the three extrinsic WOC polypeptides while minimizing damage to the apo-PSII protein and, importantly, eliminating the need to use reductants. Second, an ultrasensitive, fast-response, polarographic cell and detection system were built. The apparatus features (a) an ultrabright red light-emitted diode (LED) for controlling the light intensity, pulse duration, and dark intervals, features critical for minimization of photoinhibition; (b) a microvolume (5 mu L) O-2 polarographic cell (Clark type) fitted with a thin silicone membrane for rapid response (100 ms); and (c) DC/AC preamplifier integrated into the microcell and interfaced to a bandpass AC amplifier. The sensitivity enables detection of similar to 5 x 10(-14) mol of O-2 per flash at a signal to noise = 5/1. These improvements permit 100-fold lower Mn concentrations to be explored. Under optimum conditions, complete recovery of O-2-evolving activity could be restored compared to that of PSII membranes depleted of the three extrinsic polypeptides (35% V-max vs intact PSII). Titration of the photoactivation steady-state O-2 yield, Y-ss, and the half-time for recovery, t(1/2), vs Mn concentration demonstrate that 4.0 Mn/P680 are cooperatively taken up at 95% restoration of Y-ss and that 1.1-1.2 Mn atoms are involved in the rate-limiting photolytic step under steady-state conditions. Due to minimization of photoihibition, this intermediate exhibits a single exponential recovery kinetic over the entire population of PSII centers. Mn atoms in excess of 4 Mn/P680 accelerate the rate of photoactivation but decrease the yield above 8-10 Mn/P680. Maxima in both Y-ss and t(1/2) are observed at similar electrochemical potentials of the medium, 380 and 340 mV, respectively. We attribute this maximum to either elimination of a recombination reaction between the redox-active tyrosine-161 of the D1 polypeptide (Y-z(+)) and an electron acceptor, possibly cytochrome b(559), or stabilization of an intermediate in photoactivation. At low Mn2+ concentrations, a new pre-steady-state kinetic intermediate which binds fewer than 4 Mn atoms can be directly observed. This early kinetic phase has a rate that depends on Mn concentration and is independent of the electron acceptor identity and concentration.