title: Spectroscopic and crystallographic characterization of "alternative resting" and "resting oxidized" enzyme forms of bilirubin oxidase: implications for activity and electrochemical behavior of multicopper oxidases.
abstract: While there is broad agreement on the catalytic mechanism of multicopper oxidases (MCOs), the geometric and electronic structures of the resting trinuclear Cu cluster have been variable, and their relevance to catalysis has been debated. Here, we present a spectroscopic characterization, complemented by crystallographic data, of two resting forms occurring in the same enzyme and define their interconversion. The resting oxidized form shows similar features to the resting form in Rhus vernicifera and Trametes versicolor laccase, characterized by "normal" type 2 Cu electron paramagnetic resonance (EPR) features, 330 nm absorption shoulder, and a short type 3 (T3) Cu-Cu distance, while the alternative resting form shows unusually small A(||) and high g(||) EPR features, lack of 330 nm absorption intensity, and a long T3 Cu-Cu distance. These different forms are evaluated with respect to activation for catalysis, and it is shown that the alternative resting form can only be activated by low-potential reduction, in contrast to the resting oxidized form which is activated via type 1 Cu at high potential. This difference in activity is correlated to differences in redox states of the two forms and highlights the requirement for efficient sequential reduction of resting MCOs for their involvement in catalysis.
authors.affiliation: Department of Chemistry, Stanford University, Stanford, California 94305, United States.
authors.forename: Christian H
authors.initials: CH
authors.lastname: Kjaergaard
authors.forename: Fabien
authors.initials: F
authors.lastname: Durand
authors.forename: Federico
authors.initials: F
authors.lastname: Tasca
authors.forename: Munzarin F
authors.initials: MF
authors.lastname: Qayyum
authors.forename: Brice
authors.initials: B
authors.lastname: Kauffmann
authors.forename: Sébastien
authors.initials: S
authors.lastname: Gounel
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Suraniti
authors.forename: Keith O
authors.initials: KO
authors.lastname: Hodgson
authors.forename: Britt
authors.initials: B
authors.lastname: Hedman
authors.forename: Nicolas
authors.initials: N
authors.lastname: Mano
authors.forename: Edward I
authors.initials: EI
authors.lastname: Solomon
mesh_terms: D020082:Magnaporthe / Q000201:enzymology*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry* / Q000378:metabolism; D029045:Rhus / Q000201:enzymology*; D055454:Trametes / Q000201:enzymology*; D056928:X-Ray Absorption Spectroscopy
chemical_list: D044925:Oxidoreductases Acting on CH-CH Group Donors; C042401:bilirubin oxidase