Simulation of the cavity-binding site of three bacterial multicopper oxidases upon complex stabilization: interactional profile and electron transference pathways

Abstract

Previous studies have shown that multicopper oxidases (MCOs) oxidize organic and inorganic compounds through oxidation-reduction reactions in which three structurally and functionally arranged copper centers coordinate the uptake of an electron from a reduced substrate. Structural comparisons among three bacterial MCOs, with high structural homology and available three-dimensional information, reveal that the primary structural differences between these MCOs are located near the mononuclear copper center (T1Cu), where substrate oxidation occurs, as opposed to where the reduction of oxygen to water occurs at the trinuclear center. Nevertheless, this substrate oxidation is achieved through an outer-sphere electron transfer mechanism that does not generate a stable substrate-enzyme complex. In this study, MCOs from Thermus thermophilus (Tth-MCO), Bacillus subtilis (CotA), and Escherichia coli (CueO), which have been previously determined through X-ray crystallography, were used as models to analyze the binding modes of these MCOs to three organic molecules, with specific interest in the substrate-binding site. The binding mode of the electron-donor molecule to the electron transfer binding site was primarily attributed to hydrophobic contacts, which likely play an important role in the determination of substrate specificity. Some complexes generated in this study showed an electron donor molecule conformation in which an electron could be directly transferred to the histidines coordinating T1Cu, while for others additional electron transference pathways were also possible through the participation of charged residues during electron transfer.

Interplay between Orientation at Electrodes and Copper Activation of Thermus thermophilus Laccase for O2 Reduction

“…low affinity. Interestingly, MD simulations of Tt Lac and analogous hairpin-deletant earlier demonstrated a similar manner of ABTS docking to both molecules despite hairpin absence thus confirming our experimental observations. , …”

“…Interestingly, MD simulations of Tt Lac and analogous hairpin-deletant earlier demonstrated a similar manner of ABTS docking to both molecules despite hairpin absence thus confirming our experimental observations. 54,55 In agreement with the solution assay, the Δ-hairpin Lac behaved similarly to the wild type when immobilized on Neg-CNTs or Pos-CNTs (Figure S16). On Neg-CNTs no DET process could be observed, and a catalytic wave similar to the WT was obtained in the presence of Cu 2+ .…”

Mutations in the coordination spheres of T1 Cu affect Cu2+-activation of the laccase from Thermus thermophilus

“…The carbon nanofibers used in the current work present a hydrophobic surface, suggesting that hydrophobic interaction should also play a key role in the efficient electrical wiring of the enzyme [35]. This is not unexpected as it was demonstrated that substrate-enzyme complex was mainly driven by hydrophobic interactions [43]. The mutants were then adsorbed on the same carbon nanofiber-modified electrode.…”

Distal Mutations Shape Substrate-Binding Sites during Evolution of a Metallo-Oxidase into a Laccase

“…Both cavities are comparable to those in other prokaryotic MCOs (Figure S3): cavity A is closer to the substrate-binding site in Bacillus subtilis CotA and cavity B to the substrate-binding site in E. coli CueO and Thermus thermophilus Tth multicopper oxidases …”