Pogni Rebecca, Baratto M Camilla, Giansanti Stefania, Teutloff Christian, Verdin Jorge, Valderrama Brenda, Lendzian Friedhelm, Lubitz Wolfgang, Vazquez-Duhalt Rafael, Basosi Riccardo
Department of Chemistry, University of Siena, via Aldo Moro, 53100 Siena, Italy.
Biochemistry. 2005 Mar 22;44(11):4267-74. doi: 10.1021/bi047474l.
Versatile peroxidase (VP) from Bjerkandera adusta is a structural hybrid between lignin (LiP) and manganese (MnP) peroxidase. This hybrid combines the catalytic properties of the two above peroxidases, being able to oxidize typical LiP and MnP substrates. The catalytic mechanism is that of classical peroxidases, where the substrate oxidation is carried out by a two-electron multistep reaction at the expense of hydrogen peroxide. Elucidation of the structures of intermediates in this process is crucial for understanding the mechanism of substrate oxidation. In this work, the reaction of H(2)O(2) with the enzyme in the absence of substrate has been investigated with electron paramagnetic resonance (EPR) spectroscopy. The results reveal an EPR signal with partially resolved hyperfine structure typical of an organic radical. The yield of this radical is approximately 30%. Progressive microwave power saturation measurements indicate that the radical is weakly coupled to a paramagnetic metal ion, suggesting an amino acid radical in moderate distance from the ferryl heme. A tryptophan radical was identified as a protein-based radical formed during the catalytic mechanism of VP from Bjerkandera adusta through X-band and high-field EPR measurements at 94 GHz, aided by computer simulations for both frequency bands. A close analysis of the theoretical model of the VP from Bjerkandera sp. shows the presence of a tryptophan residue near to the heme prosthetic group, which is solvent-exposed as in the case of LiP and other VPs. The catalytic role of this residue in a long-range electron-transfer pathway is discussed.
来自烟管菌的多功能过氧化物酶（VP）是木质素过氧化物酶（LiP）和锰过氧化物酶（MnP）之间的结构杂合体。这种杂合体结合了上述两种过氧化物酶的催化特性，能够氧化典型的LiP和MnP底物。其催化机制与经典过氧化物酶相同，即底物氧化通过以过氧化氢为代价的双电子多步反应进行。阐明该过程中中间体的结构对于理解底物氧化机制至关重要。在这项工作中，利用电子顺磁共振（EPR）光谱研究了在无底物情况下H₂O₂与该酶的反应。结果揭示了一个具有部分分辨超精细结构的EPR信号，这是有机自由基的典型特征。该自由基的产率约为30%。逐步微波功率饱和测量表明，该自由基与顺磁性金属离子弱耦合，表明是一个与高铁血红素距离适中的氨基酸自由基。通过在94 GHz下进行X波段和高场EPR测量，并借助两个频段的计算机模拟，鉴定出一个色氨酸自由基是烟管菌VP催化机制中形成的基于蛋白质的自由基。对烟管菌属VP理论模型的仔细分析表明，在血红素辅基附近存在一个色氨酸残基，它像LiP和其他VP一样暴露于溶剂中。讨论了该残基在长程电子转移途径中的催化作用。
