Sakamoto Hiroshi, Takahashi Kenichi, Higashimoto Yuichiro, Harada Saori, Palmer Graham, Noguchi Masato
Department of Medical Biochemistry, Kurume University School of Medicine, Kurume, Japan.
Biochem Biophys Res Commun. 2005 Dec 9;338(1):578-83. doi: 10.1016/j.bbrc.2005.08.176. Epub 2005 Aug 31.
O2-dependent reactions of the ferric and ferrous forms of alpha-hydroxyheme complexed with water-soluble rat heme oxygenase-1 were examined by rapid-scan stopped-flow measurements. Ferric alpha-hydroxyheme reacted with O2 to form ferric verdoheme with an O2-dependent rate constant of 4x10(5) M(-1) s(-1) at pH 7.4 and 9.0. A decrease of the rate constant to 2.8x10(5) M(-1) s(-1) at pH 6.5 indicates that the reaction proceeds by direct attack of O2 on the pi-neutral radical form of alpha-hydroxyheme, which is generated by deprotonation of the alpha-hydroxy group. The reaction of ferrous alpha-hydroxyheme with O2 yielded ferrous verdoheme in a biphasic fashion involving a new intermediate having absorption maxima at 415 and 815 nm. The rate constants for this two-step reaction were 68 and 145 s(-1). These results show that conversion of alpha-hydroxyheme to verdoheme is much faster than the reduction of coordinated iron (<1 s(-1)) under physiological conditions [Y. Liu, P.R. Ortiz de Montellano, Reaction intermediates and single turnover rate constants for the oxidation of heme by human heme oxygenase-1, J. Biol. Chem. 275 (2000) 5297-5307], suggesting that, in vivo, the conversion of ferric alpha-hydroxyheme to ferric verdoheme precedes the reduction of ferric alpha-hydroxyheme.
通过快速扫描停流测量法研究了与水溶性大鼠血红素加氧酶-1复合的α-羟基血红素的三价铁和二价铁形式的氧依赖性反应。在pH 7.4和9.0条件下，三价铁α-羟基血红素与氧气反应生成三价铁胆绿素，氧依赖性速率常数为4×10⁵ M⁻¹ s⁻¹。在pH 6.5时，速率常数降至2.8×10⁵ M⁻¹ s⁻¹，这表明该反应是通过氧气直接攻击α-羟基血红素的π-中性自由基形式进行的，该自由基形式是由α-羟基的去质子化产生的。二价铁α-羟基血红素与氧气的反应以双相方式产生二价铁胆绿素，涉及一种在415和815 nm处有吸收最大值的新中间体。该两步反应的速率常数分别为68和145 s⁻¹。这些结果表明，在生理条件下，α-羟基血红素向胆绿素的转化比配位铁的还原（<1 s⁻¹）快得多[Y. Liu, P.R. Ortiz de Montellano, 人血红素加氧酶-1氧化血红素的反应中间体和单周转速率常数, J. Biol. Chem. 275 (2000) 5297-5307]，这表明在体内，三价铁α-羟基血红素向三价铁胆绿素的转化先于三价铁α-羟基血红素的还原。
