Ke B, Hansen R E, Beinert H
Charles F. Kettering Research Laboratory, Yellow Springs, Ohio, 45387.
Proc Natl Acad Sci U S A. 1973 Oct;70(10):2941-5. doi: 10.1073/pnas.70.10.2941.
Digitonin - fractionated photosystem - I subchloroplasts were titrated potentiometrically between -450 and -610 mV at pH 10. Examination of the titrated subchloroplasts by low-temperature (13 degrees K) electron paramagnetic resonance spectroscopy revealed resonances centered at values of 2.05, 1.94, 1.92, 1.89, and 1.86 on the g-factor scale. The peak heights depended on the potentials at which the chloroplasts were poised. The resonances of at least three iron-sulfur centers can be recognized: one with lines at g = 2.05 and 1.94; one with lines at g = 2.05, 1.92, and 1.89; and one for which only a line at g = 1.86 has been resolved. The midpoint potentials of the iron-sulfur species fall into two distinctly separate regions: the titration profile of the g = 1.94 signal, the first segment of the g = 2.05 plot, and the rise phase of the g = 1.86 signal had a value of -530 +/- 5 mV; the upper segment of the g = 2.05 plot, the decrease phase of the g = 1.86 signal, and the g = 1.89 profile had a midpoint potential estimated to be [unk] -580 mV. The oxidation-reduction reaction of each of the bound iron-sulfur species, as represented by the changes of the electron paramagnetic resonance spectra, was reversible and apparently involved a two-electron change.Titration at pH 9 could only be carried to -560 mV, and essentially only the first half of the titration behavior as found at pH 10 was seen. At any given potential more positive than -560 mV, the part of the iron-sulfur protein that was not reduced electrochemically could be reduced photochemically, but only to the maximum extent reduced electrochemically at -560 mV. Whereas, chloroplasts illuminated at room temperature and then frozen while still being illuminated developed a signal similar to that produced by electrochemical reduction at -610 mV, illumination at 77 degrees K did not bring about photoreduction beyond that accomplished electrochemically at about -560 mV.Dithionite alone in the dark and under anaerobic conditions brought about a partial reduction to the extent of the first electrochemical reduction step. Dithionite plus illumination at room temperature or dithionite plus methyl viologen in the dark produced the maximum signal. Electron paramagnetic resonance spectra due to either light or electrochemically reduced iron-sulfur proteins showed no detectable decay for at least 3 days when samples were stored in the dark at 77 degrees K.
在pH 10条件下，用电位滴定法对洋地黄皂苷分级分离的光系统I亚叶绿体进行了-450至-610 mV的滴定。通过低温（13 K）电子顺磁共振光谱对滴定后的亚叶绿体进行检测，发现在g因子标度上共振峰集中在2.05、1.94、1.92、1.89和1.86处。峰高取决于叶绿体所处的电位。至少可以识别出三个铁硫中心的共振峰：一个在g = 2.05和1.94处有谱线；一个在g = 2.05、1.92和1.89处有谱线；还有一个仅在g = 1.86处有一条已分辨的谱线。铁硫物种的中点电位分为两个明显不同的区域：g = 1.94信号的滴定曲线、g = 2.05曲线的第一段以及g = 1.86信号的上升阶段的中点电位值为-530±5 mV；g = 2.05曲线的上半段、g = 1.86信号的下降阶段以及g = 1.89曲线的中点电位估计为[未知] -580 mV。由电子顺磁共振光谱变化所代表的每个结合铁硫物种的氧化还原反应是可逆的，且显然涉及两电子变化。在pH 9条件下滴定只能达到-560 mV，基本上只能看到在pH 10时发现的滴定行为的前半部分。在任何给定的比-560 mV更正的电位下，未被电化学还原的铁硫蛋白部分可以被光化学还原，但只能达到在-560 mV时电化学还原的最大程度。而在室温下光照然后在仍光照时冷冻的叶绿体产生的信号类似于在-610 mV电化学还原产生的信号，在77 K下光照不会导致光还原超过在约-560 mV时电化学完成的程度。在黑暗和厌氧条件下单独使用连二亚硫酸盐会导致部分还原至第一个电化学还原步骤的程度。连二亚硫酸盐加室温光照或连二亚硫酸盐加黑暗中的甲基紫精会产生最大信号。当样品在77 K黑暗中储存时，由光或电化学还原的铁硫蛋白产生的电子顺磁共振光谱至少3天没有可检测到的衰减。
