Spectroscopic and Crystallographic Characterization of “Alternative Resting” and “Resting Oxidized” Enzyme Forms of Bilirubin Oxidase: Implications for Activity and Electrochemical Behavior of Multicopper Oxidases

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.