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 ...
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.
CueO from Escherichia coli, a member of the multi-copper oxidase (MCO) family, was examined as a direct electron transfer-type bioelectrocatalyst for the four-electron reduction of O2. Although CueO requires the fifth copper located near the type I Cu site to exhibit its oxidase activity (Roberts et al., 2003), it has been found that CueO receives electrons directly from electrodes even in the absence of the fifth copper. The fact indicates that electrons are transferred directly from electrodes to the type I Cu site. Furthermore, the catalytic current density of as large as about −4 mA cm−2 was successfully observed with a rotating pyrolytic graphite electrode at pH 5. CueO is found to be superior to other MCOs in view of the catalytic activity and is an important candidate as a catalyst of the cathode in biofuel cells.
Asp112 adjacent to the trinuclear Cu center of a multicopper oxidase, CueO was mutated for Glu, Ala and Asn. Mutations on Asp112 affected not only spectroscopic and magnetic properties derived from the trinuclear Cu center but also enzyme activities. The uncoordinated Asp112 was found to play multiple roles to promote the binding of dioxygen at the trinuclear Cu center and to accelerate the conversion of dioxygen to water molecules by facilitating the supply of H + to the reaction intermediates.
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