Evidence from spin-trapping for a transient radical on tryptophan residue 171 of lignin peroxidase

被引:57
作者
Blodig, W
Smith, AT
Winterhalter, K
Piontek, K
机构
[1] Swiss Fed Inst Technol, ETH Zurich, Biochem Lab, CH-8092 Zurich, Switzerland
[2] Univ Sussex, Sch Biol Sci, Brighton BN1 9QG, E Sussex, England
基金
英国生物技术与生命科学研究理事会;
关键词
lignin peroxidase; spin trapping; tryptophanyl radical;
D O I
10.1006/abbi.1999.1365
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The heme enzyme lignin peroxidase contains a unique C beta-hydroxylated tryptophan residue (Trp171) on the surface of the enzyme. Mutagenetic substitution of Trp171 abolishes completely the veratryl alcohol oxidation activity of the enzyme. This led us to surmise that Trp171 may be involved in electron transfer from natural substrates to the heme cofactor. Here we present evidence for the formation of a transient radical on Trp171 using spin-trapping in combination with peptide mapping. The spin-trap methyl nitroso propane forms a covalent adduct with Trp171 in the presence of hydrogen peroxide which can be detected by its characteristic visible absorbance spectrum. A very similar chromophore can be obtained in a small molecular model system from N-acetyl tryptophanamide, the spin-trap, and a single-electron abstracting system, The precise site the spin-trap is attached to could be identified in a crystal structure of spin-trap/ hydrogen peroxide-treated enzyme as the C6 atom of the indole ring of Trp171. These results indicate that Trp171 is redox-active and that it forms an indole radical by transfer of an electron to the heme of compound I and/or II. Apart from cytochrome c peroxidase and DNA photolyase, lignin peroxidase appears to be the third enzyme only which utilizes a tryptophan residue as an integral part of its redox catalysis. (C) 1999 Academic Press.
引用
收藏
页码:86 / 92
页数:7
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