Gunther M R, Tschirret-Guth R A, Witkowska H E, Fann Y C, Barr D P, Ortiz De Montellano P R, Mason R P
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233 Research Triangle Park, NC 27709, USA.
Biochem J. 1998 Mar 15;330 ( Pt 3)(Pt 3):1293-9. doi: 10.1042/bj3301293.
The reaction between metmyoglobin and hydrogen peroxide produces both a ferryl-oxo heme and a globin-centred radical(s) from the two oxidizing equivalents of the hydrogen peroxide. Evidence has been presented for localization of the globin-centred radical on one tryptophan residue and tyrosines 103 and 151. When the spin-trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) is included in the reaction mixture, a radical adduct has been detected, but the residue at which that adduct is formed has not been determined. Replacement of either tryptophans 7 and 14 or tyrosines 146 and 151 with phenylalanine has no effect on the formation of DMPO adduct in the reaction with hydrogen peroxide. When tyrosine 103 is replaced with phenylalanine, however, only DMPOX, a product of the oxidation of the spin-trap, is detected. Tyrosine-103 is, therefore, the site of radical adduct formation with DMPO. The spin trap 2-methyl-2-nitrosopropane (MNP), however, forms radical adducts with any recombinant sperm whale metmyoglobin that contains either tyrosine 103 or 151. Detailed spectral analysis of the DMPO and MNP radical adducts of isotopically substituted tyrosine radical yield complete structural determinations. The multiple sites of trapping support a model in which the unpaired electron density is spread over a number of residues in the population of metmyoglobin molecules, at least some of which are in equilibrium with each other.
高铁肌红蛋白与过氧化氢之间的反应，利用过氧化氢的两个氧化当量，生成了一个高铁-氧血红素和一个以球蛋白为中心的自由基。已有证据表明，以球蛋白为中心的自由基定位于一个色氨酸残基以及酪氨酸103和151上。当反应混合物中加入自旋捕获剂5,5-二甲基-1-吡咯啉N-氧化物（DMPO）时，检测到了一种自由基加合物，但尚未确定该加合物形成于哪个残基上。用苯丙氨酸取代色氨酸7和14或酪氨酸146和151，对与过氧化氢反应中DMPO加合物的形成没有影响。然而，当酪氨酸103被苯丙氨酸取代时，只检测到了自旋捕获剂氧化产物DMPOX。因此，酪氨酸-103是与DMPO形成自由基加合物的位点。然而，自旋捕获剂2-甲基-2-亚硝基丙烷（MNP），能与任何含有酪氨酸103或151的重组抹香鲸高铁肌红蛋白形成自由基加合物。对同位素取代酪氨酸自由基的DMPO和MNP自由基加合物进行详细的光谱分析，可完成结构测定。多个捕获位点支持这样一个模型，即未成对电子密度分布在高铁肌红蛋白分子群体中的多个残基上，其中至少有一些残基处于相互平衡状态。
