T. C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA.
Chem Biodivers. 2012 Sep;9(9):1703-17. doi: 10.1002/cbdv.201100448.
The hemoglobin from the cyanobacterium Synechococcus sp. PCC 7002 (GlbN) contains three tyrosines (Tyr5, Tyr22, and Tyr53), each of which undergoes a structural rearrangement when the protein binds an exogenous ligand such as cyanide. We explored the use of 3-fluorotyrosine and (19)F-NMR spectroscopy for the characterization of GlbN. Assignment of (19)F resonances in fluorinated GlbN (GlbN*) was achieved with individual Tyr5Phe and Tyr53Phe replacements. We observed marked variations in chemical shift and linewidth reflecting the dependence of structural and dynamic properties on oxidation state, ligation state, and covalent attachment of the heme group. The isoelectronic complexes of ferric GlbN* with cyanide and ferrous GlbN* with carbon monoxide gave contrasting spectra, the latter exhibiting heterogeneity and enhanced internal motions on a microsecond-to-millisecond time scale. The strength of the H-bond network involving Tyr22 (B10) and bound cyanide was tested at high pH. 3-Fluorotyrosine at position 22 had a pK(a) value at least 3 units higher than its intrinsic value, 8.5. In addition, evidence was found for long-range communication among the tyrosine sites. These observations demonstrated the utility of the 3-fluorotyrosine approach to gain insight in hemoglobin properties.
来自聚球藻属 PCC 7002（GlbN）的血红蛋白含有三个酪氨酸残基（Tyr5、Tyr22 和 Tyr53），当蛋白质结合外源性配体如氰化物时，每个残基都会发生结构重排。我们探索了使用 3-氟代酪氨酸和 (19)F-NMR 光谱来表征 GlbN。通过单独的 Tyr5Phe 和 Tyr53Phe 取代来实现氟化 GlbN (GlbN*)中 (19)F 共振的分配。我们观察到化学位移和线宽的明显变化，反映了结构和动态性质对氧化态、配体状态和血红素基团的共价结合的依赖性。Ferric GlbN与氰化物和 Ferrous GlbN与一氧化碳的等电子复合物给出了对比的光谱，后者在微秒至毫秒时间尺度上表现出异质性和增强的内部运动。在高 pH 值下测试了涉及 Tyr22（B10）和结合氰化物的氢键网络的强度。位置 22 的 3-氟代酪氨酸的 pK(a)值至少比其固有值 8.5 高 3 个单位。此外，还发现了酪氨酸位点之间的远程通信的证据。这些观察结果表明，3-氟代酪氨酸方法在研究血红蛋白性质方面具有实用性。
