Abstract
Unlike any protein studied so far, the active site of bilirubin oxidase from Myrothecium verrucaria contains a unique type of covalent link between tryptophan and histidine side chains. The role of this post-translational modification in substrate binding and oxidation is not sufficiently understood. Our structural and mutational studies provide evidence that this Trp396–His398 adduct modifies T1 copper coordination and is an important part of the substrate binding and oxidation site. The presence of the adduct is crucial for oxidation of substituted phenols and it substantially influences the rate of oxidation of bilirubin. Additionally, we bring the first structure of bilirubin oxidase in complex with one of its products, ferricyanide ion, interacting with the modified tryptophan side chain, Arg356 and the active site-forming loop 393-398. The results imply that structurally and chemically distinct types of substrates, including bilirubin, utilize the Trp–His adduct mainly for binding and to a smaller extent for electron transfer.
Highlights
Bilirubin oxidase (MvBOx; EC 1.3.3.5) from the ascomycete plant pathogen Myrothecium verrucaria (Albifimbria verrucaria) is a member of the blue multicopper oxidase family (MCO)
The mechanism of dioxygen reduction inside the trinuclear cluster (TNC) as well as the electron transfer path between the T1Cu site and the TNC are very similar within the MCO family and are well understood
MvBOx crystallized in the space group F222 with two monomers in the asymmetric unit (ASU)
Summary
Bilirubin oxidase (MvBOx; EC 1.3.3.5) from the ascomycete plant pathogen Myrothecium verrucaria (Albifimbria verrucaria) is a member of the blue multicopper oxidase family (MCO). One copper ion is of type I (T1Cu) and is present at the so called T1Cu site near the protein surface Coordination of this copper ion is responsible for the distinctive blue color of MvBOx and all MCOs (absorption at 600 nm) and for oxidation of substrates with the Cu2+ ion being an electron acceptor[9,10,11]. The mechanism of dioxygen reduction inside the TNC as well as the electron transfer path between the T1Cu site and the TNC are very similar within the MCO family and are well understood They were intensively studied using biochemical, structural, and computational methods[12,16,34,35,36]. We examined its role in the reaction mechanism by mutagenesis connected with structure-function analysis
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