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

Abstract

The catalytic cycle of multicopper oxidases (MCOs) involves intramolecular electron transfer (IET) from the Cu‐T1 copper ion, which is the primary site of the one‐electron oxidations of the substrate, to the trinuclear copper cluster (TNC), which is the site of the four‐electron reduction of dioxygen to water. In this study we report a detailed characterization of the kinetic and electrochemical properties of bilirubin oxidase (BOx) – a member of the MCO family. The experimental results strongly indicate that under certain conditions, e.g. in alkaline solutions, the IET can be the rate‐limiting step in the BOx catalytic cycle. The data also suggest that one of the catalytically relevant intermediates (most likely characterized by an intermediate oxidation state of the TNC) formed during the catalytic cycle of BOx has a redox potential close to 0.4 V, indicating an uphill IET process from the T1 copper site (0.7 V) to the Cu‐T23. These suggestions are supported by calculations of the IET rate, based on the experimentally observed Gibbs free energy change and theoretical estimates of reorganization energy obtained by combined quantum and molecular mechanical (QM/MM) calculations.

Fabrication of high surface area graphene electrodes with high performance towards enzymatic oxygen reduction

“…Therefore, this α value could be attributed to a mechanism where both first (outer electron transfer from the electrode) and second (intramolecular electron transfer) are rate-limiting [45]. On the one hand, these results are in excellent agreement with previously published data concerning possible changes of the rate limiting step during bioelectrocatalytic reduction of oxygen by BOx due to pH changes of the electrolyte, i.e., from heterogeneous to intramolecular ET in acidic and basic media, respectively [41,52]. On the other hand, we cannot rule out an effect of heterogeneous current density distribution due to the high surface area provided by graphene on the electrode surface [53].…”

Reorganization Energy for Internal Electron Transfer in Multicopper Oxidases

“…Single-point calculations give almost the same results as calculations with optimised structures, as is normally assumed. Experimentally, the redox potential of T1 sites with this set of ligand in various proteins is 0.3-0.7 V 33,35,89,90 (that of CueO is 0.4-0.5 V, depending on pH 91 ), showing that only the estimates obtained in a low-dielectric medium are in a reasonable range.…”

“…29,31,32 Recently, it has been suggested that the reaction mechanism of the MCOs involves an uphill intramolecular electron-transfer (IET) step. 33,34,35 Many biological system contain an uphill electron-transfer step, and it has been hypothesized that this feature exists to avoid the need of optimising individual rates. 36 This suggestion can be justified by correlating observed differences in redox potentials and IET rates.…”

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

“…Potential values for T2 and T3 centers are more difficult to evaluate because of the many intermediates with different geometries and oxidation states that are generated in the course of O 2 reduction. [47,52,[58][59][60] Some works however reported multiple noncatalytic redox waves for MCOs in the range + 0.2 to + 0.4 V vs. Ag/AgCl, most probably because of a different enzyme orientation depending on the electrode material. [47,[61][62] At least one of these processes might be attributed to a trinuclear cluster intermediate.…”

“…[41,47,[63][64] An uphill intramolecular electron transfer was thus proposed, which was confirmed both by electrochemistry and quantum and molecular mechanical calculations. [60] This process is, however, still a matter of debate because the catalytic cycle involves highly oxidative species. [34,51,57] LACs are characterized by an acidic isoelectric point and an optimal activity under acidic pH.…”