Hienerwadel R, Berthomieu C
CEA Saclay, Section de Bioénergétique, Gif-sur-Yvette, France.
Biochemistry. 1995 Dec 19;34(50):16288-97. doi: 10.1021/bi00050a008.
The binding site of the non-heme iron of photosystem II (PS II) is investigated by light-induced Fourier tranform infrared (FTIR) difference spectroscopy on Tris-washed membranes. The non-heme iron is oxidized (Fe3+) in the dark with ferricyanide and reduced (Fe2+) after light-induced charge separation by electron transfer from the semiquinone anion QA-. EPR experiments and IR modes of ferri- and ferrocyanide show that the electron donor side of PS II is reduced in less than 2 s after a flash and that ferricyanide reoxidizes the non-heme iron with a half-time of approximately 20 s. Recording FTIR spectra before and 2 s after flash illumination thus results in the Fe2+/Fe3+ difference spectrum. This spectrum shows band shifts and intensity changes of IR modes from ligands and neighboring residues of the non-heme iron. The IR modes of bicarbonate are revealed by comparison of Fe2+/Fe3+ spectra obtained on PS II membranes with 12C or 13C isotope labeled bicarbonate in H2O and in 2H2O. The nu as(CO) and nu s(CO) modes of bicarbonate in the Fe2+ state are assigned at 1530 +/- 10 and 1338 cm-1, respectively. The low frequency of the nu as(CO) mode is taken as experimental evidence that bicarbonate is a ligand of the non-heme iron. Furthermore, the small frequency difference (192 cm-1) between the nu as(CO) and nu s(CO) modes as compared to even hydrogen-bonded ionic bicarbonate strongly indicates that bicarbonate is a bidentate ligand of the non-heme iron in PS II. Upon iron oxidation, the bicarbonate modes are largely affected. The nu s(CO) mode is assigned at 1228 cm-1, while the nu as(CO) mode is tentatively assigned at 1658 +/- 20 cm-1. The strong up- and downshifts of the nu as and nu s(CO) modes of bicarbonate upon iron oxidation results in a frequency difference of 430 +/- 20 cm-1 that is not only explained by the increased charge on the iron but indicates that bicarbonate is a monodentate ligand of the oxidized iron. The sensitivity of the nu s(CO) mode of bicarbonate to 1H/2H exchange in both the Fe2+ and Fe3+ states and the presence in the Fe2+ state of a delta (COH) mode at 1258 cm-1 confirm that bicarbonate and not carbonate is the iron ligand and further exhibits hydrogen bond(s) with the protein. The 13C isotope-sensitive modes of bicarbonate are not affected by 15N labeling of the PS II membranes. 15N sensitive signals at 1111/1102 and 1094 cm-1 are assigned to side chain modes from histidine ligands of the iron. The latter signal is proposed to account for a histidine ligand that deprotonates upon iron oxidation. The involvement of protein peptide groups and side chains in the hydrogen-bond network around the iron is also discussed.
通过对Tris洗涤过的膜进行光诱导傅里叶变换红外（FTIR）差示光谱，研究了光系统II（PS II）非血红素铁的结合位点。非血红素铁在黑暗中被铁氰化物氧化为（Fe3+），在光诱导电荷分离后，通过半醌阴离子QA-的电子转移而还原为（Fe2+）。电子顺磁共振（EPR）实验以及铁氰化铁和亚铁氰化铁的红外模式表明，PS II的电子供体侧在闪光后不到2秒内被还原，并且铁氰化物以约20秒的半衰期将非血红素铁重新氧化。因此，在闪光照射前和照射后2秒记录FTIR光谱，可得到Fe2+/Fe3+差示光谱。该光谱显示了来自非血红素铁配体和相邻残基的红外模式的谱带位移和强度变化。通过比较在含有12C或13C同位素标记的碳酸氢盐的H2O和2H2O中的PS II膜上获得的Fe2+/Fe3+光谱，揭示了碳酸氢盐的红外模式。Fe2+状态下碳酸氢盐的νas(CO)和νs(CO)模式分别位于1530±10和1338 cm-1处。νas(CO)模式的低频被视为碳酸氢盐是非血红素铁配体的实验证据。此外，与甚至氢键结合的离子型碳酸氢盐相比，νas(CO)和νs(CO)模式之间的小频率差（192 cm-1）强烈表明，碳酸氢盐是PS II中非血红素铁的双齿配体。铁氧化后，碳酸氢盐模式受到很大影响。νs(CO)模式位于1228 cm-1处，而νas(CO)模式暂定为1658±20 cm-1。铁氧化时碳酸氢盐的νas和νs(CO)模式的强烈上移和下移导致频率差为430±20 cm-1，这不仅可以用铁上电荷增加来解释，还表明碳酸氢盐是氧化态铁的单齿配体。碳酸氢盐的νs(CO)模式在Fe2+和Fe3+状态下对1H/2H交换的敏感性以及在Fe2+状态下1258 cm-1处的δ(COH)模式的存在，证实了与铁结合的是碳酸氢盐而非碳酸盐，并且进一步表明其与蛋白质存在氢键。碳酸氢盐的13C同位素敏感模式不受PS II膜15N标记的影响。1111/1102和1094 cm-1处的15N敏感信号归因于铁的组氨酸配体的侧链模式。后一个信号被认为是一个在铁氧化时去质子化的组氨酸配体。还讨论了蛋白质肽基团和侧链在铁周围氢键网络中的参与情况。
