Hydrogen-1 nuclear magnetic resonance investigation of high-potential iron-sulfur proteins from Ectothiorhodospira halophila and Ectothiorhodospira vacuolata: a comparative study of hyperfine-shifted resonances.
Hydrogen-1 nuclear magnetic resonance investigation of high-potential iron-sulfur proteins from Ectothiorhodospira halophila and Ectothiorhodospira vacuolata: a comparative study of hyperfine-shifted resonances.
复制标题
嗜盐外硫红螺和空泡外硫红螺高电位铁硫蛋白的 Hydrogen-1 核磁共振研究：超精细位移共振的比较研究。
DOI：
10.1021/bi00349a010
复制
发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Meyer,TE
中科院分区：
文献类型：
--
作者：
Krishnamoorthi,R;Markley,JL;Cusanovich,MA;Przysiecki,CT;Meyer,TE
亚利桑那大学生物化学系，图森，亚利桑那州 85721 收到 1985 年 6 月 14 日摘要：高电位铁硫蛋白 (HiPIP) 的氧化和还原形式的质子 NMR 谱在 200 MHz 下记录。研究的蛋白质是来自 Ectothiorhodospira halophila 和 Ectothiorhodospira vacuolata 的 HiPIPs I 和 II。根据化学位移、纵向弛豫时间和温度依赖性行为，氧化蛋白质光谱中的超精细位移峰被分配给活性位点的半胱氨酰配体和芳香族残基的一些质子。发现半胱氨酰 C^-H 质子在低场（约 100 ppm）共振，而 Ca-H 质子在高场（约 25 ppm）共振。这种超精细位移模式与观察到的各向同性位移是起源接触的一致；它可能是a-自旋转移机制的结果。分配给芳香族残基的峰的大幅化学位移表明这些残基通过ir重叠与铁硫簇相互作用。发现一些在水中观察到的超精细位移峰在 2H2O 溶液中消失。这种共振可能是由直接与铁硫簇形成氢键的氨基酸残基的交换不稳定氢引起的。对于来自 E. vacuolata 的 HiPIPs I 和 II，除了峰的数量外，其光谱相似，推断存在于氧化和还原蛋白质中的氢键的相对数量定性地解释了它们中点氧化还原电位之间的差异。另一方面，对于 E. halophila HiPIPs I 和 II，仅考虑推断的氢键数量无法预测它们中点氧化还原电位之间差异的符号。后两种蛋白质表现出不同的不可交换超精细位移峰模式，氧化的嗜盐大肠杆菌 HiPIP II 在高场有一对额外的峰，这归因于与铁硫簇接触的芳香族残基。这种芳香族相互作用似乎可以调节这些 HiPIP 活性位点的氧化还原电位。光合细菌中发现的高电位铁硫蛋白 (HiPIP) 1 (A/r< 9000) 会发生可逆的单电子转移反应。其在生化途径中的具体功能作用尚不清楚；然而，人们发现 Chromatium vinosum HiPIP 很容易与从同一细菌中分离出的硫代硫酸盐氧化酶相互作用（Fukumari & Yamanaka，1979）。氧化HiPIP的(Cys-S-)4-Fe4S4辅基是顺磁性的，带有净负电荷；在简化形式中，它主要是反磁性的，因为簇的两个不成对的自旋是交换耦合的，导致反铁磁性（Phillips等人，1970）。铁硫蛋白研究的一个基本问题是确定给定类型活性位点氧化还原电位的调节来源。提出了以下两种机制：（i）铁硫簇的电子几何结构的多肽限制（Carter，1977b；Carter 等人，1974；Laskowski 等人，1978）
Department of Biochemistry, University of Arizona, Tucson, Arizona 85721 Received June 14, 1985 abstract: Proton NMR spectra of the oxidized and reduced forms of high-potential iron-sulfur proteins (HiPIPs) were recorded at 200 MHz. The proteins studied were the HiPIPs I and II from Ectothiorhodospira halophila and Ectothiorhodospira vacuolata. Hyperfine-shifted peaks in spectra of the oxidized proteins were assigned to some of the protons of the cysteinyl ligands and aromatic residues at the active site on the basis of their chemical shifts, longitudinal relaxation times, and temperature-dependent behavior. The cysteinyl C^-H protons were found to resonate downfield (about 100 ppm) and the Ca-H protons upfield (about-25 ppm). This hyperfine shift pattern is consistent with the observed isotropicshift being contact in origin; it probably results from a-spin-transfer mechanism. The large magnitudes of the chemical shifts of peaks assigned to aromatic residues suggest that these residues interact with the iron-sulfur cluster via-ir overlap. Some of the hyperfine-shifted peaks observed in water were found to disappear in 2H20 solution. Such resonances probably arise from exchange-labile hydrogens of amino acidresidues directly hydrogen bonded to the iron-sulfur cluster. In the case of HiPIPs I and II from E. vacuolata, whose spectra are similar except for the number of such peaks, the relative number of hydrogen bonds inferred to be present in the oxidized and reduced proteins qualitatively explains the difference between their midpoint redox potentials. On the other hand, for E. halophila HiPIPs I and II, consideration of the inferred number of hydrogen bonds alone fails to predict thesign of the difference between their midpoint redox potentials. The latter two proteins exhibited different patterns of nonexchangeable hyperfine-shifted peaks with oxidized E. halophila HiPIP II having an additional pair of peaks athigh field that were attributed to aromatic residues in contact with the iron-sulfur cluster. Such aromatic interactions appear to modulate the redox potential of the active site in these HiPIPs.High-potential iron-sulfur protein (HiPIP) 1 (A/r< 9000), found in photosynthetic bacteria, undergoes reversible one-electron-transfer reactions. Its specific functional role in biochemical pathways is unknown; however, Chromatium vinosum HiPIP has been found to interact readily with a thiosulfate-oxidizing enzyme isolated from the same bacterium (Fukumari & Yamanaka, 1979). The (Cys-S-) 4-Fe4S4 prosthetic group of oxidized HiPIP is paramagnetic with a net negative charge; in the reduced form, it is predominantly diamagnetic, because the two unpaired spins of the cluster are exchange-coupled, resulting in antiferromagnetism (Phillips et al., 1970). A fundamental issue in iron-sulfur protein research is the identification of sources of modulation of the redox potential of a given type of active site. The following two mechanisms have been suggested:(i) polypeptide con-straints of the electronic geometry of the iron-sulfur cluster (Carter, 1977b; Carter et al., 1974; Laskowski et al., 1978)