Ligand and halide binding properties of chloroperoxidase: peroxidase-type active site heme environment with cytochrome P-450 type endogenous axial ligand and spectroscopic properties.
Ligand and halide binding properties of chloroperoxidase: peroxidase-type active site heme environment with cytochrome P-450 type endogenous axial ligand and spectroscopic properties.
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氯过氧化物酶的配体和卤化物结合特性：过氧化物酶型活性位点血红素环境与细胞色素 P-450 型内源性轴向配体和光谱特性。
DOI：
10.1021/bi00350a011
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Hager,LP
中科院分区：
文献类型：
--
作者：
Sono,M;Dawson,JH;Hall,K;Hager,LP
接收于1985年7月2日的摘要：外源性配体和卤化物与氯过氧化物酶活性位点血红素铁的平衡结合研究已经在pH 2 ~ 7范围内进行了。研究了二十多种配体，包括C、N、O、P、S给体和四种卤化物。从光学吸收光谱的变化判断，在所有情况下，配体都与铁或亚铁氯过氧化物酶的血红素铁直接结合；在几种情况下，铁酶与氰化物的竞争实验证实了这一点。除了卤化物、硝酸盐和醋酸盐外，所有的配体都形成了低自旋配合物，与光谱相关蛋白细胞色素P-450-CAM的结果类似[Sono， M., & Dawson， J. H.(1982) J. Biol.]。滴定结果表明，对于铁酶，(i)弱酸性配体（pKa bbb3）以其中性（质子化）形式与酶结合，然后在连接到血红素铁时进行去质子化。相比之下，（ii）强酸性配体（pKa< 0），包括SCN“、N03”和除F"外的卤化物可能以阴离子（去质子化）形式与酶的酸形式结合：蛋白质上具有pATa< 2的单个可电离基团参与这种结合。对于含铁酶，（iii） pKa值为5.5的单个可电离基团影响配体结合。这些结果表明，尽管氯过氧化物酶在光谱和血红素铁配位结构上与P-450酶相似，但从其配体结合特性和活性位点血红素环境来看，它显然属于氢过氧化物酶。磁性圆二色性研究表明，碱性形式（pH 9.5）的铁氯过氧化物酶具有rs -铁血红素-N给体配位结构，N给体可能来源于组氨酸咪唑。氯过氧化物酶是血红素铁类酶中检测结构与功能关系的一种独特的蛋白质。对氯过氧化物酶（vide infra）的广泛研究表明，它的光谱性质与细胞色素P-450.1的光谱性质密切对应，这种相似性通常被认为是由本质上相同的血红素铁配位结构造成的。然而，就催化活性而言，氯过氧化物酶属于氢氧化酶类别，而P-450是单加氧酶。已观察到的氯过氧化物酶和P-450光谱的相似之处包括紫外可见吸收（Hollenberg & Hager, 1973）、Móssbauer （Champion等人，1973,1975）、磁圆二色性（MCD）（Dawson等人，1976；Sono等人，1984）、EPR （Hollenberg等人，1980）、共振拉曼（Champion等人，1976；Hall等人，1985）和扩展x射线吸收精细结构（Cramer等人，1978）光谱研究。特别是，这两种酶在CO与亚铁酶结合（Hollenberg & Hager, 1973）、硫酸盐配体与铁酶结合（Dawson et al., 1983; Sono et al., 1984）和磷配体与铁或t结合时，都表现出高卟啉（分裂Soret）光谱，在450nm左右具有不寻常的红移soret峰。圆二色分光光度计的电磁铁是通过研究公司的拨款购买的。
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 Received July 2, 1985 abstract: Equilibrium binding studies of exogenous ligands and halides to the active site heme iron of chloroperoxidase havebeen carried out from pH 2 to 7. Over twenty ligandshave been studied including C, N, O, P, and S donors and the four halides. As judgedfrom changes in the optical absorption spectra, direct binding of the ligands to the heme iron of ferric or ferrous chloroperoxidase occurs in all cases; this has been ascertained for the ferric enzyme in several cases through competition experiments with cyanide. All of the ligands except for the halides, nitrate, and acetate form exclusively low-spin complexes in analogy to results obtained with the spectroscopically related protein, cytochrome P-450-CAM [Sono, M., & Dawson, J. H.(1982) J. Biol. Chem. 257, 5496-5502], The titration results show that, for the ferric enzyme,(i) weakly acidic ligands (pKa> 3) bind to the enzyme in their neutral (protonated) form, followed by de-protonation upon ligation to the heme iron. In contrast,(ii) strongly acidic ligands (pKa< 0) including SCN", N03", and the halides except for F" likely bind in their anionic (deprotonated) form to the acid form of the enzyme: a single ionizable group on the proteinwith a pATa< 2 is involved in this binding. For the ferrous enzyme,(iii) a single ionizable group with the pKa value of 5.5 affects ligand binding. These results reveal that chloroperoxidase, in spite of the previously established close spectroscopic and heme iron co-ordination structure similarities to the P-450 enzymes, clearly belongs to the hydroperoxidases in terms of its ligand binding properties and active site heme environment. Magnetic circular dichroism studies indicate that the alkaline form (pH 9.5) of ferric chloroperoxidase has an RS-ferric heme-N donor ligand coordination structure with the N donor likely derived from histidine imidazole.(chloroperoxidase is a unique protein with which to test the relationships between structure and function in the heme iron class of enzymes. Extensive studies of chloroperoxidase (vide infra) have revealed close correspondence betweenits spectral properties and those of cytochrome P-450.1 Such similarities are generally presumed to result from essentially identical heme iron coordination structures. And yet, in terms of catalytic activity, chloroperoxidase belongs to the hydroper-oxidase category of enzymes while P-450 is a monooxygenase. The spectral parallels that havebeen observed for chloro-peroxidase and P-450 have included studies with UV-visible absorption (Hollenberg & Hager, 1973), Móssbauer (Cham-pion et al., 1973, 1975), magnetic circular dichroism (MCD)(Dawson et al., 1976; Sono et al., 1984), EPR (Hollenberg et al., 1980), resonance Raman (Champion et al., 1976; Hall et al., 1985), and extended X-ray absorption fine structure (Cramer et al., 1978) spectroscopy. In particular, both en-zymes exhibit hyperporphyrin (split Soret) spectra with un-usually red-shifted Soretpeaks around 450 nm upon binding of CO to the ferrous enzyme (Hollenberg & Hager, 1973), thiolate ligands to the ferric enzyme (Dawson et al., 1983; Sono et al., 1984), and phosphine ligands to either the ferric or t Supported by Grants GM-26730 (JHD) and GM-07768 (LPH) from the US Public Health Service. The electromagnet for the circular dichroism spectrophotometer was purchased through a grant from Research Corporation.