Tyrosinase Model Complexes using Imidazole Ligands

描述(申请人提供)：自然界中许多蛋白质的活性部位都含有铜，这些部位通常与氧化还原催化有关，具有广泛的生物效应。 功能如电子载体蛋白(天青素、质蓝蛋白)、加氧酶活性(酪氨酸酶、 甲烷单加氧酶、半乳糖氧化酶、环色素C氧化酶)和氧运输(血蓝蛋白)。具体地说，3型铜位包含两个紧密相连的铜离子，每个铜离子通过三个组氨酸氮连接到蛋白质骨架上。这些类型-3的铜中心能够可逆地结合氧气(O2)，以传输氧气以及氧化有机和无机底物。类型3铜中心中的一个特别重要的酶是酪氨酸酶。在哺乳动物中，这种酶参与了黑色素形成的生物合成途径。该酶催化羟化，随后酪氨酸双电子氧化为多巴奎酮。鉴于哺乳动物对酪氨酸酶的这种酶功能，该酶与皮肤色素异常有关，如斑点和缺陷。在最近的研究中，酪氨酸酶被认为与帕金森病和其他神经退行性疾病有关。因此，酪氨酸酶在医药、农业和工业中具有重要意义。鉴于酪氨酸酶的重要酶功能，尽管其密切的反应细节仍不清楚，但已有重要的研究。通常认为，这一机理是通过m-H_2：H_2-过氧二铜(II)物种(SP)发生的。虽然铜可以以铜(I)、铜(II)和铜(III)的氧化态存在，但铜(III)通常不被认为具有生物相关性，因为它具有相当的正电性 氧化还原电位(约500 mV vs.Se)，尽管这些想法早于铜(I)胺络合物与O2的简单反应中铜(III)的表征。最近的研究表明，底物结合诱导重排为异构体双间氧代二铜(III)中间体(O)，该中间体执行苯酚氧化，其电子和动力学同位素参数与酶值一致。然而，这些研究和该领域的大多数其他研究都使用了非生物相关的配体，如吡啶、吡嗪或烷基保护的咪唑。这一建议旨在通过计算研究和光谱研究在模型络合物中使用更多具有生物相关性的配体来探索铜(III)氧化态的生物相关性。这些关于铜(III)生物相关性的发现将提供铜生物化学(例如，非血红素铁中心的Fe(IV)-氧中间体)的完全范式转变。 公共卫生相关性：这项建议旨在了解铜(III)在3型铜酶中的生物学相关性。为了解决这个问题，将使用模型络合物进行计算研究、配体设计、极端溶液温度(-125℃)光谱和反应性研究。具体地说，该建议强调使用含有N-H键的螯合咪唑配体来探索咪唑到咪唑的转变如何影响铜(I)与铜(II)与铜(III)模型配合物的相对稳定性。

DESCRIPTION (provided by applicant): Many proteins in nature contain copper in their active sites, and these sites are typically involved in redox catalysis performing a wide array biological functions such as electron carrier proteins (azurin, plastocyanin), oxygenase activity (tyrosinase, methane monooxygenase, galactose oxidase, cyctochrome c oxidase), and oxygen transport (hemocyanin). Specifically, type-3 copper sites contain 2 copper ions in close proximity and each copper is bonded to the protein backbone by three histidine nitrogens. These type-3 copper centers are capable of reversibly binding dioxygen (O2) for the purposes of dioxygen transport and the oxidation of organic and inorganic substrates. One especially important enzyme in the class of type-3 copper centers is tyrosinase. In mammals, the enzyme is involved in the biosynthetic pathway for the formation of melanin. The enzyme catalyzes the hydroxylation followed by a two electron oxidation of tyrosine to dopaquinone. Given this mammalian enzymatic function for tyrosinase, the enzyme has been linked to skin pigmentation abnormalities such as flecks and defects. In recent studies, the tyrosine enzyme has been linked to Parkinson disease and other neurodegenerative disease. As such, the tyrosinase enzyme is quite significant in medicine, agriculture, and industry. Given the important enzymatic function of tyrosinase, significant study has occurred although the intimate reaction details are still unknown. Generally, the mechanism is considered to occur through a m-h2:h2- peroxo-dicopper(II) species (SP). Although copper can exist in oxidation states of Cu(I), Cu(II), and Cu(III), Cu(III) is generally not considered to be biologically relevant due to its rather positive redox potential (ca. 500 mV vs. SHE), though these ideas preceded the characterization of Cu(III) in simple reactions of Cu(I) amine complexes with O2. Recent studies have implicated a substrate-binding induced rearrangement to an isomeric bis-m-oxo-dicopper(III) intermediate (O) that performs phenol oxidation with electronic and kinetic isotope parameters consistent with enzymatic values. However, these and most other studies in the field have used non-biologically relevant ligands such as pyridine, pyrazine, or alkyl protected imidazoles. This proposal aims to use ligands more biologically relevant ligands in model complexes through computational studies and spectroscopic investigations to probe the biological relevance of the Cu(III) oxidation state. Such findings of the biological relevance of Cu(III) would provide a complete paradigm shift in copper biochemistry (e.g. Fe(IV)-oxo intermediates for non-heme iron sites). PUBLIC HEALTH RELEVANCE: This proposal aims to gain understanding of the biological relevance of Cu(III) in type-3 copper enzymes. To address this issue, computational studies, ligand design, and extreme solution temperature (- 125¿C) spectroscopy and reactivity studies will be performed using model complexes. Specifically, the proposal stresses the use of chelating imidazole ligands containing N-H bonds to probe how imidazole to imidazolate transformation affect the relative stability in Cu(I) versus Cu(II) versus Cu(III) model complexes.