Emerson Joseph P, Kovaleva Elena G, Farquhar Erik R, Lipscomb John D, Que Lawrence
Department of Chemistry, Center for Metals in Biocatalysis, University of Minnesota, Minneapolis, MN 55455, USA.
Proc Natl Acad Sci U S A. 2008 May 27;105(21):7347-52. doi: 10.1073/pnas.0711179105. Epub 2008 May 20.
Biological O(2) activation often occurs after binding to a reduced metal [e.g., M(II)] in an enzyme active site. Subsequent M(II)-to-O(2) electron transfer results in a reactive M(III)-superoxo species. For the extradiol aromatic ring-cleaving dioxygenases, we have proposed a different model where an electron is transferred from substrate to O(2) via the M(II) center to which they are both bound, thereby obviating the need for an integral change in metal redox state. This model is tested by using homoprotocatechuate 2,3-dioxygenases from Brevibacterium fuscum (Fe-HPCD) and Arthrobacter globiformis (Mn-MndD) that share high sequence identity and very similar structures. Despite these similarities, Fe-HPCD binds Fe(II) whereas Mn-MndD incorporates Mn(II). Methods are described to incorporate the nonphysiological metal into each enzyme (Mn-HPCD and Fe-MndD). The x-ray crystal structure of Mn-HPCD at 1.7 A is found to be indistinguishable from that of Fe-HPCD, while EPR studies show that the Mn(II) sites of Mn-MndD and Mn-HPCD, and the Fe(II) sites of the NO complexes of Fe-HPCD and Fe-MndD, are very similar. The uniform metal site structures of these enzymes suggest that extradiol dioxygenases cannot differentially compensate for the 0.7-V gap in the redox potentials of free iron and manganese. Nonetheless, all four enzymes exhibit nearly the same K(M) and V(max) values. These enzymes constitute an unusual pair of metallo-oxygenases that remain fully active after a metal swap, implicating a different way by which metals are used to promote oxygen activation without an integral change in metal redox state.
生物性氧气激活通常在氧气与酶活性位点中的还原态金属（如M(II)）结合后发生。随后的M(II)到O₂的电子转移会产生一种具有反应活性的M(III)-超氧物种。对于间位二醇芳香环裂解双加氧酶，我们提出了一种不同的模型，即电子通过它们共同结合的M(II)中心从底物转移到O₂，从而避免了金属氧化还原状态发生整体变化的需要。通过使用来自短褐杆菌的原儿茶酸2,3-双加氧酶（Fe-HPCD）和球形节杆菌的原儿茶酸2,3-双加氧酶（Mn-MndD）对该模型进行了测试，这两种酶具有高度的序列同一性和非常相似的结构。尽管有这些相似之处，但Fe-HPCD结合Fe(II)，而Mn-MndD结合Mn(II)。文中描述了将非生理性金属掺入每种酶（Mn-HPCD和Fe-MndD）的方法。发现1.7 Å分辨率下的Mn-HPCD的X射线晶体结构与Fe-HPCD的无法区分，而电子顺磁共振研究表明，Mn-MndD和Mn-HPCD的Mn(II)位点，以及Fe-HPCD和Fe-MndD的NO配合物的Fe(II)位点非常相似。这些酶的金属位点结构一致，表明间位二醇双加氧酶无法差异补偿游离铁和锰的氧化还原电位中0.7 V的差距。尽管如此，所有这四种酶都表现出几乎相同的K(M)和V(max)值。这些酶构成了一对不同寻常的金属加氧酶，在金属交换后仍保持完全活性，这意味着金属用于促进氧气激活的方式不同，且金属氧化还原状态没有整体变化。
