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Rapid mixing apparatus for detection of Fe(I) intermediate(s) of hemoproteins with emphasis on P-450

用于检测血红素蛋白 Fe(I) 中间体的快速混合装置，重点关注 P-450

基本信息

批准号：
60870012
负责人：
ORII Yutaka
金额：
$ 7.68万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research
财政年份：
1985
资助国家：
日本
起止时间：
1985 至 1987
项目状态：
已结题

项目摘要

Heme catabolism in an important biological process, which is caralyzed by the enzyme heme oxygenase. P-450 is involved in the hydroxylation of steroids, alkanes and in the metabolism of various drugs. <alpha>-Oxprotoheme, in early intermediate in heme catabolism, was synthesized and its autoxidation to biliverdin IX<alpha> was studied using rapid mixing sampling apparatus. The apparatus was prepared for mixing two different samples anaerobically or aerobically below -10゜C, followed by recording electronic spectra and ESR spectra. The experiment demonstrate severel lives of evidence for a unique electronic structure of <alpha>-oxyprotoheme, which is a resonance hybrid between Fe(II)-oxyprotoporphyrin <pi>-neutral radical and Fe(III)-oxyprotorphyrin anion. The data for Fe(III)-oxyprotoporphyrin in basic solvent are consistent with the fromulation of Fe(II)-oxyprotorphyrin <pi>-neutral radical, in which the hydroxy proton is deprotonated to afford theenolate from, therby electron transfer occurring from the enolate anion to the ferric iron to form the Fe(II)-oxyprotoporphyrin <pi>-neutral radical. Such resonance structures depends on the ligand basicity. It is interesting to note that the ESR spectrum of Fe(II)-oxyprotoporphyrin <pi>-neutral radical in basic solvent such as 4-acetylpyridine is quite similar to that for Fe(I)-protoporphyrin which lead us to propose in our prevoius report that cooling to 77K causes one more electron transfer from the porphyrin to Fe(II) to form at Fe(I) compound. In P-450 model system (protoheme-n-butylmercaptide), however, ESR spectrum was silent and showed no Fe(i) spectra which demonstrated one electron transfer from S^- to Fe(II). Instead high oxidation state of Fe(IV) was demonstrated.Using a rapid mixing sampling apparatus, the novel 893 nm intermediate was detected with halflife time of 300 ms during the conversion of <alpha>-oxyprotoheme to <alpha>-versohemochrome.

血红素加氧酶是血红素分解的重要生物过程。P-450参与类固醇、烷烃的羟基化和各种药物的代谢。<alpha>合成了血红素催化剂的早期中间体-氧化血红素，并利用快速混合进样装置研究了它对胆绿素IX的自氧化作用<alpha>。该装置用于在低于-10 ° C的厌氧或需氧条件下混合两种不同的样品，然后记录电子光谱和ESR光谱。实验证明<alpha>了Fe（Ⅱ）-氧化原卟啉<pi>-中性自由基与Fe（Ⅲ）-氧化原卟啉阴离子之间的共振杂化物- Fe（Ⅲ）-氧原卟啉在碱性溶剂中的数据与Fe（Ⅱ）-氧原卟啉中性自由基的公式一致<pi>，其中羟基质子被去质子化，形成烯醇化物，烯醇化物阴离子与三价铁离子发生电子转移，形成Fe（Ⅱ）-氧原卟啉<pi>中性自由基。这种共振结构取决于配体的碱性。有趣的是，Fe（II）-氧原卟啉<pi>-中性自由基在碱性溶剂如4-乙酰基吡啶中的ESR谱与Fe（I）-原卟啉的ESR谱非常相似，这使我们在先前的报告中提出，冷却到77 K会导致从卟啉到Fe（II）的多一个电子转移，形成Fe（I）化合物。而在P-450模型体系（血红素-正丁基硫醇盐）中，ESR谱没有显示Fe（i）谱，表明S^-向Fe（II）发生了一个电子转移。用快速混合进样装置检测到-氧化血红素转化为-versohemochrome过程中的一个新的893 nm的中间体，半衰期为300 ms<alpha><alpha>。