Vassiliev I R, Jung Y S, Yang F, Golbeck J H
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park 16802, USA.
Biophys J. 1998 Apr;74(4):2029-35. doi: 10.1016/S0006-3495(98)77909-3.
The PsaC subunit of photosystem I (PS I) binds two [4Fe-4S] clusters, F(A) and F(B), functioning as electron carriers between F(X) and soluble ferredoxin. To resolve the issue whether F(A) or F(B) is proximal to F(X), we used single-turnover flashes to promote step-by-step electron transfer between electron carriers in control (both F(A) and F(B) present) and HgCl2-treated (F(B)-less) PS I complexes from Synechococcus sp. PCC 6301 and analyzed the kinetics of P700+ reduction by monitoring the absorbance changes at 832 nm in the presence of a fast electron donor (phenazine methosulfate (PMS)). In control PS I complexes exogenously added ferredoxin, or flavodoxin could be photoreduced on each flash, thus allowing P700+ to be reduced from PMS. In F(B)-less complexes, both in the presence and in the absence of ferredoxin or flavodoxin, P700+ was reduced from PMS only on the first flash and was reduced from F(X)- on the following flashes, indicating lack of electron transfer to ferredoxin or flavodoxin. In the F(B)-less complexes, a normal level of P700 photooxidation was detected accompanied by a high yield of charge recombination between P700+ and F(A)- in the presence of a slow donor, 2,6-dichlorophenol-indophenol. This recombination remained the only pathway of F(A)- reoxidation in the presence of added ferredoxin, consistent with the lack of forward electron transfer. F(A)- could be reoxidized by methyl viologen in F(B)-less PS I complexes, although at a concentration two orders of magnitude higher than is required in wild-type PS I complexes, thus implying the presence of a diffusion barrier. The inhibition of electron transfer to ferredoxin and flavodoxin was completely reversed after reconstituting the F(B) cluster. Using rate versus distance estimates for electron transfer rates from F(X) to ferredoxin for two possible orientations of PsaC, we conclude that the kinetic data are best compatible with PsaC being oriented with F(A) as the cluster proximal to F(X) and F(B) as the distal cluster that donates electrons to ferredoxin.
光系统I（PS I）的PsaC亚基结合两个[4Fe-4S]簇，即F(A)和F(B)，它们作为F(X)与可溶性铁氧化还原蛋白之间的电子载体发挥作用。为了解决F(A)或F(B)哪个更靠近F(X)的问题，我们使用单周转闪光来促进来自集胞藻属PCC 6301的对照（同时存在F(A)和F(B)）和HgCl2处理（不含F(B)）的PS I复合物中电子载体之间的逐步电子转移，并通过在快速电子供体（硫酸甲基吩嗪（PMS））存在下监测832 nm处的吸光度变化来分析P700+还原的动力学。在对照PS I复合物中，外源添加的铁氧化还原蛋白或黄素氧化还原蛋白在每次闪光时都能被光还原，从而使P700+能从PMS得到还原。在不含F(B)的复合物中，无论是否存在铁氧化还原蛋白或黄素氧化还原蛋白，P700+仅在第一次闪光时从PMS得到还原，而在随后的闪光中从F(X)-得到还原，这表明缺乏向铁氧化还原蛋白或黄素氧化还原蛋白的电子转移。在不含F(B)的复合物中，在存在缓慢供体2,6-二氯酚靛酚的情况下，检测到正常水平的P700光氧化，并伴随着P700+与F(A)-之间的高电荷复合产率。在添加铁氧化还原蛋白的情况下，这种复合仍然是F(A)-再氧化的唯一途径，这与缺乏正向电子转移一致。在不含F(B)的PS I复合物中，F(A)-可以被甲基紫精再氧化，尽管其浓度比野生型PS I复合物所需的浓度高两个数量级，因此这意味着存在扩散屏障。在重建F(B)簇后，对铁氧化还原蛋白和黄素氧化还原蛋白的电子转移抑制完全逆转。利用针对PsaC的两种可能取向从F(X)到铁氧化还原蛋白的电子转移速率的速率与距离估计，我们得出结论，动力学数据与PsaC的取向最相符，即F(A)作为靠近F(X)的簇，F(B)作为向铁氧化还原蛋白供电子的远端簇。
