Role of a Tyrosine Radical in Human Ceruloplasmin Catalysis.

Multicopper oxidases (MCOs) are a large family of diverse enzymes found in both eukaryotes and prokaryotes that couple one-electron oxidations of various substrates to the four-electron reduction of O to HO, functioning through a set of metallocofactors consisting of one type 1 copper (T1 Cu) and one trinuclear copper cluster (TNC). Human serum ceruloplasmin (Cp) is a unique member of MCOs composed of six cupredoxin domains and harbors six Cu ions arranged as three T1 Cu and one TNC. The native substrate of Cp is Fe. It is an essential ferroxidase critical for iron homeostasis and is closely associated with metal-mediated diseases and metal neurotoxicity. In human serum, Cp operates under substrate-limiting low [Fe] but high [O] conditions, implying the possible involvement of partially reduced intermediates in Cp catalysis. In this work, we studied for the first time Cp reactivities at defined partially reduced states and discovered a tyrosine radical weakly magnetically coupled to the native intermediate (NI) of the TNC via a hydrogen bond. Our results lead to a new hypothesis that human iron transport is regulated as the paired transfer of iron from ferroportin to Cp to transferrin, and the tyrosine residue in Cp acts as a gate to avoid reactive oxygen species (ROS) formation when Fe delivery is dysregulated.