Salvato B, Santamaria M, Beltramini M, Alzuet G, Casella L
Department of Biology, CNR Center for the Biochemistry and Physiology of Metalloproteins, University of Padova, Italy.
Biochemistry. 1998 Oct 6;37(40):14065-77. doi: 10.1021/bi980879j.
Hemocyanin and tyrosinase are dinuclear copper proteins capable of reversibly binding dioxygen. Despite the great similarity of structure and properties of their active site, the two proteins perform different biological functions (oxygen transport/storage versus monooxygenase and oxidase activity). In this paper, we show that Octopus vulgaris hemocyanin exhibits a tyrosinase-like activity; namely, it is capable of utilizing dioxygen for the oxidation of o-diphenol to quinone. The reaction is specific for this isomer of diphenol, the meta and para isomers being unreactive, and is strongly controlled by steric factors. Dioxygen represents a cosubstrate of the reaction, and it is involved in the catalytic turnover by binding to the dinuclear copper site of the protein to form, under steady-state conditions, oxy-Hc, which is the active species. The generation of semiquinone radicals, detected by EPR and by their reaction with N,N,N',N'-tetramethyl-1,4-phenylenediamine, strongly supports a reaction mechanism in which such radicals represent the reaction products of one-electron oxidation of the substrate, quinone being generated by dismutation of semiquinones. Met-Hc is regenerated by the substrate to the deoxy form. To close the catalytic cycle, the proposed reaction mechanism also involves the participation of two transient protein forms with the total oxidation state of the active site (V and IV) intermediate between that of oxy-Hcy, [CuIIO22-CuII]VI, and deoxy-Hc, [CuICuI]II. A mathematical model has been elaborated to describe the reaction kinetics. The differences in reaction mechanisms between hemocyanin and tyrosinase are discussed in terms of accessibility to exogenous molecules of their active sites.
血蓝蛋白和酪氨酸酶是能够可逆结合双原子氧的双核铜蛋白。尽管它们活性位点的结构和性质极为相似，但这两种蛋白执行不同的生物学功能（氧运输/储存与单加氧酶和氧化酶活性）。在本文中，我们表明普通章鱼血蓝蛋白具有类似酪氨酸酶的活性；也就是说，它能够利用双原子氧将邻二酚氧化为醌。该反应对二酚的这种异构体具有特异性，间位和对位异构体无反应，并且受到空间因素的强烈控制。双原子氧是该反应的共底物，它通过与蛋白质的双核铜位点结合参与催化周转，在稳态条件下形成氧合血蓝蛋白（oxy-Hc），这是活性物种。通过电子顺磁共振（EPR）检测到的半醌自由基及其与N，N，N'，N'-四甲基-1,4-苯二胺的反应，有力地支持了一种反应机制，即这些自由基代表底物单电子氧化的反应产物，醌由半醌的歧化反应生成。高铁血蓝蛋白（Met-Hc）由底物再生为脱氧形式。为了完成催化循环，所提出的反应机制还涉及两种具有活性位点总氧化态（V和IV）的瞬时蛋白形式的参与，其介于氧合血蓝蛋白（oxy-Hcy，[CuIIO22-CuII]VI）和脱氧血蓝蛋白（deoxy-Hc，[CuICuI]II）之间。已精心构建了一个数学模型来描述反应动力学。从活性位点对外源分子的可及性方面讨论了血蓝蛋白和酪氨酸酶之间反应机制的差异。
