The functional roles of the three copper sites associated with the methionine-rich insert in the multicopper oxidase CueO from E. coli

Abstract

CueO from Escherichia coli is a multicopper oxidase (MCO) involved in copper tolerance under aerobic conditions. It features the four typical copper atoms that act as electron transfer (T1) and dioxygen reduction (T2, T3; trinuclear) sites. In addition, it displays a methionine- and histidine-rich insert that includes a helix that blocks physical access to the T1 site. In crystalline form, the insert provides at least three additional Met-rich Cu(I) binding sites Cu5 (sCu), Cu6 and Cu7 that are proposed to facilitate rapid oxidation of bound Cu(I) to Cu(II) (S. K. Singh, et al., J. Biol. Chem., 2011, 43, 37849–37857). The activities of variants featuring mutations at sites Cu5 (D360M, M355LD360N), Cu6 (M358,362S), Cu7 (M364,368S) and Cu6,7 (M358,362,364,368S) were compared to that of the wild type form using three different air-stable model substrates (2,6-dimethoxyphenol, [Cu^I(Bca)₂]³⁻ and Cu^ICu^II-PcoC, a periplasmic Cu(I) binding protein from E. coli). The results demonstrate that the three copper sites play related but distinct roles in CueO oxidase activities. The internal Cu5 site is part of the essential electron transfer pathway connecting surface-exposed sites Cu6 and Cu7 to site T1. Both Cu6 and Cu7 are dominant substrate-docking-oxidation (SDO) sites on the protein surface. However, under physiologically relevant conditions, the SDO function of Cu6 relies largely on an electron transfer pathway via Cu7 to Cu5. These Met-rich sites in CueO provide a robust cuprous oxidase function for control of Cu(I) toxicity.