Vergara Alessandro, Franzese Marisa, Merlino Antonello, Vitagliano Luigi, Verde Cinzia, di Prisco Guido, Lee H Caroline, Peisach Jack, Mazzarella Lelio
Department of Chemistry, University of Naples Federico II, Complesso Universitario Monte S. Angelo, I-80126 Naples, Italy.
Biophys J. 2007 Oct 15;93(8):2822-9. doi: 10.1529/biophysj.107.105700. Epub 2007 Jun 1.
Spontaneous autoxidation of tetrameric Hbs leads to the formation of Fe (III) forms, whose physiological role is not fully understood. Here we report structural characterization by EPR of the oxidized states of tetrameric Hbs isolated from the Antarctic fish species Trematomus bernacchii, Trematomus newnesi, and Gymnodraco acuticeps, as well as the x-ray crystal structure of oxidized Trematomus bernacchii Hb, redetermined at high resolution. The oxidation of these Hbs leads to formation of states that were not usually detected in previous analyses of tetrameric Hbs. In addition to the commonly found aquo-met and hydroxy-met species, EPR analyses show that two distinct hemichromes coexist at physiological pH, referred to as hemichromes I and II, respectively. Together with the high-resolution crystal structure (1.5 A) of T. bernacchii and a survey of data available for other heme proteins, hemichrome I was assigned by x-ray crystallography and by EPR as a bis-His complex with a distorted geometry, whereas hemichrome II is a less constrained (cytochrome b5-like) bis-His complex. In four of the five Antartic fish Hbs examined, hemichrome I is the major form. EPR shows that for HbCTn, the amount of hemichrome I is substantially reduced. In addition, the concomitant presence of a penta-coordinated high-spin Fe (III) species, to our knowledge never reported before for a wild-type tetrameric Hb, was detected. A molecular modeling investigation demonstrates that the presence of the bulkier Ile in position 67beta in HbCTn in place of Val as in the other four Hbs impairs the formation of hemichrome I, thus favoring the formation of the ferric penta-coordinated species. Altogether the data show that ferric states commonly associated with monomeric and dimeric Hbs are also found in tetrameric Hbs.
四聚体血红蛋白的自发自氧化会导致形成铁（III）形式，其生理作用尚未完全明确。在此，我们报告了通过电子顺磁共振（EPR）对从南极鱼类伯氏南极鱼、纽氏南极鱼和尖吻裸头鱼中分离出的四聚体血红蛋白氧化态的结构表征，以及对氧化的伯氏南极鱼血红蛋白进行高分辨率重新测定的X射线晶体结构。这些血红蛋白的氧化导致形成了在以往四聚体血红蛋白分析中通常未检测到的状态。除了常见的水合高铁和羟基高铁物种外，EPR分析表明，在生理pH值下，两种不同的半高铁血红素共存，分别称为半高铁血红素I和II。结合伯氏南极鱼的高分辨率晶体结构（1.5埃）以及对其他血红素蛋白现有数据的调查，通过X射线晶体学和EPR将半高铁血红素I确定为一种几何结构扭曲的双组氨酸复合物，而半高铁血红素II是一种限制较少（细胞色素b5样）的双组氨酸复合物。在所检测的五种南极鱼类血红蛋白中，有四种的半高铁血红素I是主要形式。EPR显示，对于南极鱼血红蛋白CTn，半高铁血红素I的含量大幅降低。此外，还检测到一种五配位高自旋铁（III）物种的同时存在，据我们所知，这在野生型四聚体血红蛋白中从未有过报道。分子模型研究表明，南极鱼血红蛋白CTn的67β位存在比其他四种血红蛋白中更大的异亮氨酸，取代了缬氨酸，这会损害半高铁血红素I的形成，从而有利于形成铁的五配位物种。总体而言，数据表明，与单体和二聚体血红蛋白常见的高铁状态在四聚体血红蛋白中也存在。
