Ananyev G M, Dismukes G C
Hoyt Laboratory, Department of Chemistry, Princeton University, New Jersey 08544, USA.
Biochemistry. 1996 Apr 2;35(13):4102-9. doi: 10.1021/bi952667h.
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'-tetrapropionate-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 microL) O2 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 approximately 5 x 10(-14) mol of O2 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 O2-evolving activity could be restored compared to that of PSII membranes depleted of the three extrinsic polypeptides (35% Vmax vs intact PSII). Titration of the photoactivation steady-state O2 yield, Yss, and the half-time for recovery, t1/2, vs Mn concentration demonstrate that 4.0 Mn/P680 are cooperatively taken up at 95% restoration of Yss 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 Yss and t1/2 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 b559, 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.
利用方法上的两项重大改进，对光激活过程，即光系统II（PSII）膜水氧化复合物（WOC）的组装进行了研究。首先，使用了一种新型亲脂螯合剂N，N，N'，N'-四丙酸-1，3-双（氨甲基）苯（TPDBA），它能完全提取锰和钙以及三种外在WOC多肽，同时将对脱辅基PSII蛋白的损伤降至最低，并且重要的是，无需使用还原剂。其次，构建了一个超灵敏、快速响应的极谱池和检测系统。该仪器具有以下特点：（a）一个超亮红色发光二极管（LED），用于控制光强度、脉冲持续时间和暗间隔，这些对于将光抑制降至最低至关重要；（b）一个微体积（5微升）的O2极谱池（克拉克型），配有薄硅胶膜以实现快速响应（100毫秒）；（c）集成在微池中的直流/交流前置放大器，并与带通交流放大器相连。其灵敏度能够在信噪比 = 5/1的情况下，检测到每次闪光约5×10⁻¹⁴摩尔的O2。这些改进使得能够探索低100倍的锰浓度。在最佳条件下，与去除三种外在多肽的PSII膜相比，O2释放活性能够完全恢复（35%的最大反应速率Vmax，而完整的PSII为Vmax）。光激活稳态O2产量Yss和恢复半衰期t1/2与锰浓度的滴定表明，在Yss恢复95%时，每P680有4.0个锰协同结合，并且在稳态条件下，1.1 - 1.2个锰原子参与限速光解步骤。由于光抑制降至最低，这种中间体在整个PSII中心群体中呈现单一指数恢复动力学。超过每P680 4个锰原子会加速光激活速率，但在超过8 - 10个锰/ P680时会降低产量。在介质的相似电化学电位380和340 mV下，分别观察到Yss和t1/2的最大值。我们将此最大值归因于D1多肽的氧化还原活性酪氨酸-161（Y(Z)+）与电子受体（可能是细胞色素b559）之间重组反应的消除，或者光激活中间体的稳定。在低锰离子浓度下，可以直接观察到一种结合少于4个锰原子的新的预稳态动力学中间体。这个早期动力学阶段的速率取决于锰浓度，并且与电子受体的身份和浓度无关。
