Bilirubin oxidase from Myrothecium verrucaria: X-ray determination of the complete crystal structure and a rational surface modification for enhanced electrocatalytic O2 reduction

Abstract

The blue multi-copper oxidase bilirubin oxidase (BOx) from the ascomycete plant pathogen Myrothecium verrucaria (Mv) efficiently catalyses the oxidation of bilirubin to biliverdin, with the concomitant reduction of O(2) to water, a reaction of considerable interest for low-temperature bio-fuel cell applications. We have solved the complete X-ray determined structure of Mv BOx at 2.4 Å resolution, using molecular replacement with the Spore Coat Protein A (CotA) enzyme from Bacillus subtilis (PDB code 1GSK) as a template. The structure reveals an unusual environment around the blue type 1 copper (T1 Cu) that includes two non-coordinating hydrophilic amino acids, asparagine and threonine. The presence of a long, narrow and hydrophilic pocket near the T1 Cu suggests that structure of the substrate-binding site is dynamically determined in vivo. We show that the interaction between the binding pocket of Mv BOx and its highly conjugated natural organic substrate, bilirubin, can be used to stabilise the enzyme on a pyrolytic graphite electrode, more than doubling its electrocatalytic activity relative to the current obtained by simple adsorption of the protein to the carbon surface.

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

“…An efficient modification involved reproducing the lock-and-key process between BOD and its substrate, bilirubin, at the electrode. [161,175] The same idea lead to the design of substrate analogues for Mv BOD immobilization in CNT network. [176] From the DET/MET ratio, it was evaluated that 90 % of BOD was oriented with the T1 facing the electrode.…”

“…[160] In contrast to LACs, the binding pocket in BODs is mainly hydrophilic. [42,161] Fast DET between Hases and an electrode will only be achieved if the distal FeS cluster is at a tunneling distance from the electrode. In periplasmic O 2 -sensitive [NiFe]-Hases, an anionic patch of amino acid residues, mainly glutamate residues, surrounds the FeS distal cluster.…”

Direct enzymatic bioelectrocatalysis: differentiating between myth and reality

“…Since its conception in 1978 where DET between a multicopper oxidase (MCO) (laccase) and a carbon electrode afforded enzymatic O 2 reduction, DET has evolved into an established ET pathway for many oxidoreductases, most commonly (but not limited to) laccase, bilirubin oxidase (BOx), nitrate reductase, hydrogenase and fructose dehydrogenase [31][32][33][34].…”

“…Typically, polyaromatic hydrocarbons (anthracene, naphthalene and derivatives thereof ) are the most commonly employed orientational agents [40 -44]. In the case of BOx, efficient DET can be afforded by immobilization onto multi-walled carbon nanotubes, using charged multi-walled carbon nanotubes, immobilized polyaromatic hydrocarbons and even natural substrates (bilirubin and its metabolites) [33,[45][46][47][48][49].…”

On the Possibility of Uphill Intramolecular Electron Transfer in Multicopper Oxidases: Electrochemical and Quantum Chemical Study of Bilirubin Oxidase

“…1, left part). BOx has been widely used for a long time in clinical analysis [30,31] and biofuel cells [32][33][34][35][36], but the crystal structure was published only recently [37,38]. It is a highly similar to structures of other MCO and BOx contains a full complement of four copper ions per monomer, located in domains 1 and 3 [37,38].…”