T.C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA.
Departamento de Química Inorgánica, Analítica y Química Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Instituto de Química Física de los Materiales, Medio Ambiente y Energía (INQUIMAE)-CONICET, C1428EGA Buenos Aires, Argentina.
J Inorg Biochem. 2021 Jun;219:111437. doi: 10.1016/j.jinorgbio.2021.111437. Epub 2021 Mar 24.
THB1 is a monomeric truncated hemoglobin (TrHb) found in the cytoplasm of the green alga Chlamydomonas reinhardtii. The canonical heme coordination scheme in hemoglobins is a proximal histidine ligand and an open distal site. In THB1, the latter site is occupied by Lys53, which is likely to facilitate Fe(II)/Fe(III) redox cycling but hinders dioxygen binding, two features inherent to the NO dioxygenase activity of the protein. TrHb surveys show that a lysine at a position aligning with Lys53 is an insufficient determinant of coordination, and in this study, we sought to identify factors controlling lysine affinity for the heme iron. We solved the "Lys-off" X-ray structure of THB1, represented by the cyanide adduct of the Fe(III) protein, and hypothesized that interactions that differ between the known "Lys-on" structure and the Lys-off structure participate in the control of Lys53 affinity for the heme iron. We applied an experimental approach (site-directed mutagenesis, heme modification, pH titrations in the Fe(III) and Fe(II) states) and a computational approach (MD simulations in the Fe(II) state) to assess the role of heme propionate-protein interactions, distal helix capping, and the composition of the distal pocket. All THB1 modifications resulted in a weakening of lysine affinity and affected the coupling between Lys53 proton binding and heme redox potential. The results supported the importance of specific heme peripheral interactions for the pH stability of iron coordination and the ability of the protein to undergo redox reactions.
THB1 是一种单体截短血红蛋白（TrHb），存在于绿藻莱茵衣藻的细胞质中。血红蛋白中典型的血红素配位方案是一个近端组氨酸配体和一个开放的远端位点。在 THB1 中，后者位点被 Lys53 占据，这可能有利于 Fe(II)/Fe(III)氧化还原循环，但阻碍了氧的结合，这是该蛋白的 NO 加氧酶活性所固有的两个特征。TrHb 调查表明，与 Lys53 对齐的位置上的赖氨酸是配位的不足决定因素，在本研究中，我们试图确定控制赖氨酸对血红素铁亲和力的因素。我们解决了“Lys-off”THB1 的 X 射线结构，由 Fe(III)蛋白的氰化物加合物表示，并假设在已知的“Lys-on”结构和 Lys-off 结构之间存在差异的相互作用参与控制 Lys53 对血红素铁的亲和力。我们应用了一种实验方法（定点突变、血红素修饰、Fe(III)和 Fe(II)状态下的 pH 滴定）和一种计算方法（Fe(II)状态下的 MD 模拟）来评估血红素丙酸酯-蛋白相互作用、远端螺旋帽和远端口袋组成的作用。所有 THB1 修饰都导致赖氨酸亲和力减弱，并影响 Lys53 质子结合和血红素氧化还原电位之间的偶联。结果支持特定血红素外围相互作用对于铁配位的 pH 稳定性以及蛋白质进行氧化还原反应的能力的重要性。
