Hiner A N, Martínez J I, Arnao M B, Acosta M, Turner D D, Lloyd Raven E, Rodríguez-López J N
Departamento de Biología Vegetal (Fisiología Vegetal), Facultad de Biología, Universidad de Murcia, Spain.
Eur J Biochem. 2001 May;268(10):3091-8. doi: 10.1046/j.1432-1327.2001.02208.x.
The reactivity of recombinant pea cytosolic ascorbate peroxidase (rAPX) towards H2O2, the nature of the intermediates and the products of the reaction have been examined using UV/visible and EPR spectroscopies together with HPLC. Compound I of rAPX, generated by reaction of rAPX with 1 molar equivalent of H2O2, contains a porphyrin pi-cation radical. This species is unstable and, in the absence of reducing substrate, decays within 60 s to a second species, compound I*, that has a UV/visible spectrum [lambda(max) (nm) = 414, 527, 558 and 350 (sh)] similar, but not identical, to those of both horseradish peroxidase compound II and cytochrome c peroxidase compound I. Small but systematic differences were observed in the UV/visible spectra of compound I* and authentic rAPX compound II, generated by reaction of rAPX with 1 molar equivalent H2O2 in the presence of 1 molar equivalent of ascorbate [lambda(max) (nm) = 416, 527, 554, 350 (sh) and 628 (sh)]. Compound I* decays to give a 'ferric-like' species (lambda(max) = 406 nm) that is not spectroscopically identical to ferric rAPX (lambda(max) = 403 nm) with a first order rate constant, k(decay)' = (2.7 +/- 0.3) x 10(-4) s(-1). Authentic samples of compound II evolve to ferric rAPX [k(decay) = (1.1 +/- 0.2) x 10(-3) s(-1)]. Low temperature (10 K) EPR spectra are consistent with the formation of a protein-based radical, with g values for compound I* (g parallel = 2.038, g perpendicular = 2.008) close to those previously reported for the Trp191 radical in cytochrome c peroxidase (g parallel = 2.037, g perpendicular = 2.005). The EPR spectrum of rAPX compound II was essentially silent in the g = 2 region. Tryptic digestion of the 'ferric-like' rAPX followed by RP-HPLC revealed a fragment with a new absorption peak near 330 nm, consistent with the formation of a hydroxylated tryptophan residue. The results show, for the first time, that rAPX can, under certain conditions, form a protein-based radical analogous to that found in cytochrome c peroxidase. The implications of these data are discussed in the wider context of both APX catalysis and radical formation and stability in haem peroxidases.
利用紫外/可见光谱、电子顺磁共振光谱以及高效液相色谱，研究了重组豌豆胞质抗坏血酸过氧化物酶（rAPX）对过氧化氢的反应活性、反应中间体的性质以及反应产物。rAPX与1摩尔当量的过氧化氢反应生成的化合物I含有一个卟啉π-阳离子自由基。该物种不稳定，在没有还原底物的情况下，60秒内会衰变为第二种物种，即化合物I*，其紫外/可见光谱[最大吸收波长（纳米）= 414、527、558和350（肩峰）]与辣根过氧化物酶化合物II和细胞色素c过氧化物酶化合物I的光谱相似但不相同。在化合物I与真实的rAPX化合物II的紫外/可见光谱中观察到了微小但系统的差异，真实的rAPX化合物II是在1摩尔当量抗坏血酸存在下，rAPX与1摩尔当量过氧化氢反应生成的[最大吸收波长（纳米）= 416、527、554、350（肩峰）和628（肩峰）]。化合物I衰变生成一种“类铁”物种（最大吸收波长 = 406纳米），其光谱与铁rAPX（最大吸收波长 = 403纳米）不同，一级衰变速率常数k(decay)' = (2.7 ± 0.3) × 10⁻⁴ s⁻¹。化合物II的真实样品演变为铁rAPX [k(decay) = (1.1 ± 0.2) × 10⁻³ s⁻¹]。低温（10K）电子顺磁共振光谱与基于蛋白质的自由基形成一致，化合物I* 的g值（g平行 = 2.038，g垂直 = 2.008）接近先前报道的细胞色素c过氧化物酶中色氨酸191自由基的g值（g平行 = 2.037，g垂直 = 2.005）。rAPX化合物II的电子顺磁共振光谱在g = 2区域基本无信号。对“类铁”rAPX进行胰蛋白酶消化，然后进行反相高效液相色谱分析，发现一个在330纳米附近有新吸收峰的片段，这与羟基化色氨酸残基的形成一致。结果首次表明，rAPX在某些条件下可以形成一种类似于细胞色素c过氧化物酶中发现的基于蛋白质的自由基。在APX催化以及血红素过氧化物酶中自由基形成和稳定性的更广泛背景下讨论了这些数据的意义。
