We have combined the information obtained from rapid-scan electronic absorption spectrophotometry and multifrequency (9-295 GHz) electron paramagnetic resonance (EPR) spectroscopy to unequivocally determine the electronic nature of the intermediates in milk lactoperoxidase as a function of pH and to monitor their reactivity with organic substrates selected by their different accessibilities to the heme site. The aim was to address the question of the putative catalytic role of the protein-based radicals. This experimental approach allowed us to discriminate between the protein-based radical intermediates and [Fe(IV)=O] species, as well as to directly detect the oxidation products by EPR. The advantageous resolution of the g anisotropy of the Tyr (*) EPR spectrum at high fields showed that the tyrosine of the [Fe(IV)=O Tyr (*)] intermediate has an electropositive and pH-dependent microenvironment [g(x) value of 2.0077(0) at pH >or= 8.0 and 2.0066(2) at 4.0
译文
：我们结合了从快速扫描电子吸收分光光度法和多频（9-295 GHz）电子顺磁共振（EPR）光谱学获得的信息，明确确定了牛奶乳过氧化物酶中中间体的电子性质与pH的关系，并对其进行监测与有机底物的反应性取决于它们对血红素位点的不同可及性。目的是解决基于蛋白质的自由基的假定催化作用的问题。这种实验方法使我们能够区分基于蛋白质的自由基中间体和[Fe（IV）= O]种类，并通过EPR直接检测氧化产物。高电场下Tyr（*）EPR谱的g各向异性的有利分辨率表明[Fe（IV）= O Tyr（*）]中间体的酪氨酸具有电正性和pH依赖的微环境[g（x） pH>或= 8.0时的2.0077（0）值和4.0 <或= pH <或7.5]时的2.0066（2）值可能与自由基的稳定性和功能有关。两种类型的有机分子（小的芳香族和较大的底物）使我们能够区分出不同的底物氧化机理。 [Fe（IV）= O Por（*）]是通过血红素边缘反应的苯氧异羟肟酸，邻苯二甲胺和邻氨基苯胺的氧化物种，是通过长距离电子转移（支持pH值为8）的米托蒽醌的氧化物种不涉及酪氨酰自由基，后者的形成与底物在pH 5时的氧化竞争。相反，与ABTS在pH 5时的非常有效的反应与[Fe（IV）= O Tyr（*）]是氧化性物质相一致。因此，通过X射线晶体学鉴定ABTS结合位点可能是合理药物设计中的有价值的工具。
