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 debated. Here, we present a spectroscopic characterization, complimented 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 EPR features, 330nm absorption shoulder, and a short type 3 (T3) Cu-Cu distance, while the alternative resting form shows unusually small A-parallel and high g-parallel EPR features, lack of 330nm 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 the 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.

Features and applications of bilirubin oxidases

“…1, highlighting the four histidine-rich copper-binding domains characteristic of MCOs. Spectroscopic features of BODs resemble those of other MCOs in general (Durand et al 2012a;Kjaergaard et al 2012). The purified BODs have the deep blue color generally observed for multicopper oxidases.…”

Biohydrogen for a New Generation of H2/O2 Biofuel Cells: A Sustainable Energy Perspective

“…Some intermediates in the catalytic mechanism, in particular in the presence of inhibitors or under conditions of low or high potentials, are still under consideration. [36][37][38][39] The commonly accepted mechanism is depicted in Figure 2 C, in which two consecutive electronic transfers are involved from O 2 binding at the reduced trinuclear cluster, passing through a peroxy intermediate, then the native intermediate.…”

“…[65][66][67] An accurate mechanism and the precise binding site are, however, still being established, especially as a function of pH. [38,39] Furthermore, BODs are quite stable at neutral pH. [68,69] They have been identified in various fungi and bacteria, and can exhibit chloride, urate, dithiothreitol (DTT) and ethylenediaminetetraacetic acid (EDTA) tolerance.…”

“…Some of them also present thermostable properties. [38,[70][71][72][73][74] The reasons for this thermostability, although not well understood because of the absence of crystallographic structures, may originate from a more compact structure of the protein. [75] [b] [49] 0.48 (pH 7)…”

X-ray-induced catalytic active-site reduction of a multicopper oxidase: structural insights into the proton-relay mechanism and O2-reduction states

“…Nevertheless, because of the lack of sufficient electrons to form the FR enzyme, the NI necessarily decays to the RS (Augustine et al, 2010). Indeed, the RS is rather common in a large number of crystallographic structures of MCOs deposited in the PDB (~15 MCO structures), since the catalytic cycle for O 2 reduction in the crystalline state using the protons and electrons released by X-rays is rather inefficient (Hakulinen et al, 2006;Kjaergaard et al, 2012). Thus, since the FR enzyme is the first state of the catalytic cycle for O 2 reduction of MCOs (Augustine et al, 2010), and reacts immediately with O 2 to form the PI, the structural stabilization of the O 2 state (Fig.…”