One-compartment biofuel cells without separators have been constructed, in which d-fructose dehydrogenase (FDH) from Gluconobacter sp. and laccase from Trametes sp. (TsLAC) work as catalysts of direct electron transfer (DET)-type bioelectrocatalysis in the two-electron oxidation of d-fructose and four-electron reduction of dioxygen as fuels, respectively. FDH adsorbs strongly and stably on Ketjen black (KB) particles that have been modified on carbon papers (CP) and produces the catalytic current with the maximum density of about 4 mA cm(-2) without mediators at pH 5. The catalytic wave of the d-fructose oxidation is controlled by the enzyme kinetics. The location and the shape of the catalytic waves suggest strongly that the electron is directly transferred to the KB particles from the heme c site in FDH, of which the formal potential has been determined to be 39 mV vs. Ag|AgCl|sat. KCl. Electrochemistry of three kinds of multi-copper oxidases has also been investigated and TsLAC has been selected as the best one of the DET-type bioelectrocatalyst for the four-electron reduction of dioxygen in view of the thermodynamics and kinetics at pH 5. In the DET-type bioelectrocatalysis, the electron from electrodes seems to be transferred to the type I copper site of multi-copper oxidases. TsLAC adsorbed on carbon aerogel (CG) particles with an average pore size of 22 nm, that have been modified on CP electrodes, produces the catalytic reduction current of dioxygen with a density of about 4 mA cm(-2), which is governed by the mass transfer of the dissolved dioxygen. The FDH-adsorbed KB-modified CP electrodes and the TsLAC-adsorbed CG-modified CP electrodes have been combined to construct one-compartment biofuel cells without separators. The open-circuit voltage was 790 mV. The maximum current density of 2.8 mA cm(-2) and the maximum power density of 850 microW cm(-2) have been achieved at 410 mV of the cell voltage under stirring.

CueO is a multicopper oxidase involved in the homeostasis of Cu in Escherichia coli, and functions as the sole cupric oxidase ever found. Differing from other multicopper oxidases, the substratebinding site of CueO is deeply buried under a methionine-rich helical region including α-helices 5, 6, and 7 that interfere the access of organic substrates. We deleted this region, Pro357-His406 and replaced it with a Gly-Gly linker. Crystal structures of the truncated mutant in the presence and absence of excess Cu(II) indicated that the scaffold of the CueO molecule and the metal binding sites were reserved in comparison with those of CueO. In addition, the high thermostability of the protein molecule and spectroscopic and magnetic properties due to the four Cu centers were also conserved after the truncation. As for functions, the cuprous oxidase activity of the mutant was reduced to ca.10% of that of recombinant CueO owning to the decrease in the affinity of the labile Cu site for Cu (I) ions, although activities for laccase substrates such as 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), p-phenylenediamine, and 2,6-dimethoxyphenol increased due to the changes in accessibilities of these organic substrates towards the type I Cu site. The present engineering of CueO indicates that the methionine-rich α-helices function as a barrier to interfere with the access of bulky organic substrates to provide CueO with the specificity as cuprous oxidase.Keywords: CueO; multicopper oxidase; homeostasis; truncated mutant; X-ray crystal structure A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT3 IntroductionMulticopper oxidases (MCOs) are enzymes containing a multiple copper center to catalyze the oxidation of a variety of substrates such as polyphenols, aromatic polyamines, L-ascorbate, and metal ions concomitantly with the four-electron reduction of dioxygen to water. [1][2][3][4][5] Laccase, the largest subfamily of MCOs, shows multiple functions including lignin degradation, pigmentation, and pathogenesis in fungi as well as lignin biosynthesis and wound healing in plants. [6][7][8][9][10] Therefore, structures and functions of new MCOs such as Escherichia coli CueO (formerly called YacK) [11][12][13] andBacillus subtilis CotA, 14,15 have been discussed in comparison with those of laccase.CueO is a 53.4-kDa periplasmic protein involved in the Cu efflux system, together with CopA, the P-type ATPase. 16 CueO is responsible for the oxidation of cuprous ion to less toxic cupric ion and the oxidation of enterobactin to prevent the copper-catalyzed Fenton reaction so as not to sequester iron from the environment. 17,18 CueO also catalyzes the oxidation of organic compounds including 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), p-phenylenediamine (p-PD), and 2,6-dimethoxyphenol (2,6-DMP). Oxidase activities of CueO toward these substrates are considerably low, but are fairly enhanced in the presence of an excess Cu(II) ions. 11,12 That is, the enzymatic activity of CueO is regulated by Cu ions in ...

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Bioelectrocatalytic reduction of O2 into water was archived at diffusion‐controlled rate by using enzymes (laccase from Trametes sp. and bilirubin oxidase from Myrothecium verrucaria, which belong to the family of multi‐copper oxidase) adsorbed on mesoporous carbon aerogel particle without a mediator. The current density was predominantly controlled by the diffusion of dissolved O2 in rotating‐disk electrode experiments, and reached a value as large as 10 mA cm–2 at 1 atm O2, 25 °C, and 8,000 rpm on the laccase‐adsorbed electrode. The overpotential of the bioelectrocatalytic reduction of O2 was 0.4–0.55 V smaller than that observed on a Pt disk electrode. Without any optimization, the laccase‐adsorbed biocathode showed stable current intensity of the O2 reduction in an air‐saturated buffer at least for 10 days under continuous flow system.

CueO, a multi‐copper oxidase (MCO) occurring in Escherichia coli, catalyses a four‐electron reduction of O2 in a direct electron transfer (DET) mechanism with very high electrocatalytic activity on carbon aerogel electrodes. However, the overpotential of CueO is greater than that in other MCOs. By understanding the redox properties of CueO, we attempted to reduce this overpotential. Direct electrochemistry of CueO on carbon aerogel electrodes showed a pair of redox waves derived from the type I (T1) Cu site with a redox potential ($E {^{\circ \prime} \atop {\rm T1}} $) of 0.28 V versus Ag|AgCl at pH 5.0. Dependence of $E {^{\circ \prime} \atop {\rm T1}} $ on pH suggests the participation of proton transfer and acid–base equilibrium of some amino acid residue. The shape of the catalytic current is consistent with the T1 site being an inlet of electrons in the DET bioelectrocatalysis of O2, in which case the overpotential could be reduced by shifting $E {^{\circ \prime} \atop {\rm T1}} $ towards the positive potential. To achieve this, we created mutants of CueO at M510, which is the axial ligand of the T1 Cu, and at D439, which forms a hydrogen bond with His443 coordinated with the T1 Cu. Two mutants, M510L and D439A, successfully reduced the overpotential.