Modification of Photosystem I reaction center by extraction-reconstitution of pigment cofactors

1. 通过醚处理，PsaA/B PSI复合物中的大部分Chl α被去除，而P700没有损失，得到Chl α/P700比值为12的无天线PSI RC复合物。与磷脂酰甘油一起加入Chl b后，每个P700中有9 - 14个Chl b结合到Chl α -缺失复合物的结合位点上，并且结合的Chl b与原位Chl α 2一样有效地起天线作用。用含水醚处理PSI配合物提取PSI电子输运co因子(s)的PSI RC飞秒瞬态吸收光谱结果表明，在P700-中性条件下，在630 nm激发下，还原态Ao与激发态P700的比值随着温度的降低而降低。在9k时，最多40%的RC可以经历一次电荷分离，即使在生理温度下，大约100% PSI的RC显示亚皮秒的一次电荷分离。结果表明，不完全PSI rc的电荷分离是一个较为正常的过程。为了尝试构建Zn- chl α而不是Mg-Chl，我们在高浓度Zn^<2+>中培养小球藻细胞。Zn大于0.5 mmol/1对小球藻在光照条件下自养生长具有致死作用。而异养生长的小球藻，即使在锌浓度为67 mmol/1的情况下，也能在较长的滞后期增殖。锌接种细胞在生长初期呈现出Chl的吸收光谱，峰位于415和670nm处。生长过程中出现Chl形态，在680 nm处出现峰值。锌接种细胞中chl的鉴定正在进行中。以Chl α/P700比为10的菠菜P700复合物为原料，在pH11.5、10℃条件下培养，建立了选择性降解P700和A_o的方法。pH和温度越高，P100和A_o的降解越快。当pH值降至8时，降解立即停止，但没有恢复。P700的吸收峰暂时移至670 nm后消失。P700和/或A_o的重构目前正在进行中。少

1. Most Chl α in the PsaA/B PSI complexes was removed without any loss of P700 by ether treatment, yielding the antenna-depleted PSI RC complexes that have a Chl α/P700 ratio of 12. On addition of Chl b with a phosphatidylglycerol, 9 - 14 Chl b per P700 bound to the Chl α - binding sites in the antenna-depleted complexes and the bound Chl b acted as antenna as efficiently as in situ Chl α.2. A femtosecond transient absorption spectroscopy of the PSI RC in which one (or more?) of the PSI electron-transport co factor (s) was extracted by the treatment of PSI complex with water-containing ether, showed that under the P700-neutral conditions and upon the 630 nm excitation, the ratio of the amount of the reduced Ao to that of the excited P700 decreased as the temperature goes down. At most 40 % of the RC can undergo the primary charge separation at 9 K, even though about 100 % PSI RC shows subpicosecond primary charge separation at a physiological temperature. The results suggest that a the … More rmal process is involved in the charge separation of the incomplete PSI RC.3. In order to try to construct Zn-Chl α instead of Mg-Chl, Chlorella cells were cultured at the high concentration of Zn^<2+>. Zn of more than 0.5 mmol/1 was lethal to Chlorella when grown autotrophically in the light. However, when grown heterotropphically in the dark, Chlorella had an ability to proliferate after long lag phase even in the presence of 67 mmol/1 of Zn. The Zn-inoculated cells showed the absorption spectrum of Chl with peaks at 415 and 670nm at the beginning of the growth. During the growth, there appeared a Chl form with a peak at 680 nm. Identification of the Chls in the Zn-inoculated cells is now in progress.4. The method for a selective degradation of P700 and A_o was established with spinach P700-enncned complexes that had a Chl α/P700 ratio of 10 by incubation in pH11.5 at 10 ℃. The degradation of P100 and A_o proceeded more rapid with making pH or temperature higher. The degradation stopped immediately, but did not recover, by backing pH to 8. The absorption peak of P700 temporarily shifted to 670 nm and then disappeared by this treatment. The reconstitution of P700 and/or A_o is now in progress with this preparation. Less