Characterization of pH-dependent conformational heterogeneity in Rhodospirillum rubrum cytochrome c2 using 15N and 1H NMR.
Characterization of pH-dependent conformational heterogeneity in Rhodospirillum rubrum cytochrome c2 using 15N and 1H NMR.
复制标题
使用 15N 和 1H NMR 表征红色红螺菌细胞色素 c2 中 pH 依赖性构象异质性。
DOI：
10.1021/bi00464a005
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发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Smith,GM
中科院分区：
文献类型：
--
作者：
Yu,LP;Smith,GM
用~（15）N和~ 1H NMR研究了红杜鹃（Rhodocellum rubrum）中富含~（15）N的铁细胞色素c_2的性质，发现血红素和配体t ~（15）N的~（15）N共振峰由于顺磁弛豫而增宽，使其不能被检测到。在184 ppm（pH 5.6）处明确鉴定了配体组氨酸π氮的15 N共振。单个非配体组氨酸的15 N共振仅在低pH下观察到，如在铁细胞色素中，因为互变异构化引起的严重加宽。铁细胞色素的15 N和'H光谱对pH的依赖性表明，配体组氨酸（其NH）在中性pH范围内不会解离，而是参与氢键，类似于还原状态。由于既没有去质子化，也没有非氢键形式的配体组氨酸观察到的光谱中的任一氧化态，参与生产具有不同的电子g张量的异质群体的这种形式被排除。在铁细胞色素中观察到pATa为6.2、8.6和9.2的转变。在中性pH范围内观察到Ω环周围的局部构象变化，如在铁细胞色素中。结构异质性导致血红素环甲基在位置8处的多重共振。构象之间的交换速率是温度依赖性的。pKa为6.2的跃迁归属于His-42咪唑基团。配体甲硫氨酸的位移，发生与pKa为9.2，导致总的构象变化附近的血红素中心。有多种构象在高pH值，其中只有一个是能够有效地转移电子，饱和转移实验判断。铁细胞色素的N-末端具有8.6的pKa。与其在还原状态下部分受限的迁移率相反，发现它是非常移动的，反映了铁细胞色素的松散结构
The 15N-enriched ferricytochrome c2 from Rhodospirillum rubrum has been studied by 15N and’H NMR spectroscopy as a function of pH. The 15N resonances of the heme and ligand t nitrogen are broadened beyond detection because of paramagnetic relaxation. The 15N resonance of the ligand histidine π nitrogen was unambiguously identified at 184 ppm (pH 5.6). The 15N resonances of the single nonligand histidine are observed only at low pH, as in the ferrocytochrome because of the severe broadening caused by tautomerization. The dependence of the 15N and’H spectra of the ferricytochrome on pH indicated that the ligand histidine it NH does not dissociate in the neutral pH range and is involved in a hydrogen bond, similar to that in the reduced state. Because neither deprotonated nor non-hydrogen-bonded forms of the ligand histidine are observed in the spectra of either oxidation state, the participation of such forms in producing heterogeneous populations having different electronic g tensors is ruled out. Transitions having pATa's of 6.2, 8.6, and 9.2 are observed in the ferricytochrome. The localized conformational change around the Ω loops is observed in the neutral pH range, as in the ferrocytochrome. Structural heterogeneity leads to multiple resonances of the heme ring methyl at position 8. The exchange rate between the conformations is temperature dependent. The transition with a pKa of 6.2 is assigned to the His-42 imidazole group. The displacement of the ligand methionine, which occurs with a pKa of 9.2, causes gross conformational change near the heme center. There are multiple conformations at high pH, only one of which is able to transfer electrons efficiently, as judged by saturation-transfer experiments. The N-terminus of the ferricytochrome has a pKa of 8.6. In contrast to its partially restricted mobility in the reduced state, it is found to be very mobile, reflecting a looser structure of the ferricytochrome