Smith Andrew T, Doyle Wendy A, Dorlet Pierre, Ivancich Anabella
Centre National de la Recherche Scientifique, Unité de Recherche Associée 2096 and Commissariat à l'Energie Atomique, Institut de Biologie et des Technologies Saclay, Laboratoire des Hyperfréquences, Metalloprotéines et Sytèmes de Spin, F-91191 Gif-sur-Yvette, France.
Proc Natl Acad Sci U S A. 2009 Sep 22;106(38):16084-9. doi: 10.1073/pnas.0904535106. Epub 2009 Sep 14.
The surface oxidation site (Trp-171) in lignin peroxidase (LiP) required for the reaction with veratryl alcohol a high-redox-potential (1.4 V) substrate, was engineered into Coprinus cinereus peroxidase (CiP) by introducing a Trp residue into a heme peroxidase that has similar protein fold but lacks this activity. To create the catalytic activity toward veratryl alcohol in CiP, it was necessary to reproduce the Trp site and its negatively charged microenvironment by means of a triple mutation. The resulting D179W+R258E+R272D variant was characterized by multifrequency EPR spectroscopy. The spectra unequivocally showed that a new Trp radical [g values of g(x) = 2.0035(5), g(y) = 2.0027(5), and g(z) = 2.0022(1)] was formed after the [Fe(IV)=O Por(+)] intermediate, as a result of intramolecular electron transfer between Trp-179 and the porphyrin. Also, the EPR characterization crucially showed that [Fe(IV)=O Trp-179()] was the reactive intermediate with veratryl alcohol. Accordingly, our work shows that it is necessary to take into account the physicochemical properties of the radical, fine-tuned by the microenvironment, as well as those of the preceding [Fe(IV)=O Por(+)] intermediate to engineer a catalytically competent Trp site for a given substrate. Manipulation of the microenvironment of the Trp-171 site in LiP allowed the detection by EPR spectroscopy of the Trp-171(), for which direct evidence has been missing so far. Our work also highlights the role of Trp residues as tunable redox-active cofactors for enzyme catalysis in the context of peroxidases with a unique reactivity toward recalcitrant substrates that require oxidation potentials not realized at the heme site.
木质素过氧化物酶（LiP）中与藜芦醇（一种高氧化还原电位（1.4 V）的底物）反应所需的表面氧化位点（Trp-171），通过将一个色氨酸残基引入到具有相似蛋白质折叠但缺乏该活性的灰盖鬼伞过氧化物酶（CiP）中进行改造。为了在CiP中产生对藜芦醇的催化活性，有必要通过三重突变来重现色氨酸位点及其带负电荷的微环境。所得的D179W+R258E+R272D变体通过多频电子顺磁共振光谱进行表征。光谱明确显示，在[Fe(IV)=O Por(+)]中间体之后，由于Trp-179与卟啉之间的分子内电子转移，形成了一个新的色氨酸自由基[g(x) = 2.0035(5)、g(y) = 2.0027(5)和g(z) = 2.0022(1)的g值]。此外，电子顺磁共振表征至关重要地表明，[Fe(IV)=O Trp-179()]是与藜芦醇反应的活性中间体。因此，我们的工作表明，为给定底物设计具有催化活性的色氨酸位点时，有必要考虑由微环境微调的自由基的物理化学性质，以及之前的[Fe(IV)=O Por(+)]中间体的性质。对LiP中Trp-171位点微环境的操纵使得通过电子顺磁共振光谱检测到Trp-171()成为可能，而到目前为止一直缺少其直接证据。我们的工作还强调了色氨酸残基作为可调节的氧化还原活性辅因子在过氧化物酶催化中的作用，这些过氧化物酶对需要血红素位点未实现的氧化电位的顽固底物具有独特的反应性。
