Kinetic and thermodynamic analysis of Cu²⁺-dependent reductive inactivation in direct electron transfer-type bioelectrocatalysis by copper efflux oxidase

Highlights

• Effects of Cu²⁺ on bioelectrocatalysis of copper efflux oxidase were investigated.

• Cu²⁺ electrochemically induced reductive inactivation of multicopper oxidases.

• Kinetic and thermodynamic parameters were refined using electrochemical data.

• Uncompetitive inhibition appeared reasonable for the Cu²⁺ effect on the enzyme.

• A detailed model for Cu²⁺-dependent inactivation/reactivation was proposed.

Abstract

Copper efflux oxidases (CueOs) are key enzymes in copper homeostasis systems. The mechanisms involved are however largely unknown. CueO-type enzymes share a typical structural feature composed of Methionine-rich (Met-rich) domains that are proposed to be involved in copper homeostasis. Bioelectrocatalysis using CueO-type enzymes in the presence of Cu²⁺ recently highlighted a new Cu²⁺-dependent catalytic pathway related to a cuprous oxidase activity. In this work, we further investigated the effects of Cu²⁺ on direct electron transfer (DET)-type bioelectrocatalytic reduction of O₂ by CueO at NH₂-functionalized multi-walled carbon nanotubes. The DET-type bioelectrocatalytic activity of CueO decreased at low potential in the presence of Cu²⁺, showing unique peak-shaped voltammograms that we attribute to inactivation and reactivation processes. Chronoamperometry was used to kinetically analyze these processes, and the results suggested linear free energy relationships between the inactivation/reactivation rate constant and the electrode potential. Pseudo-steady-state analysis also indicated that Cu²⁺ uncompetitively inhibited the enzymatic activity. A detailed model for the Cu²⁺-dependent reductive inactivation of CueO was proposed to explain the electrochemical data, and the related thermodynamic and kinetic parameters. A CueO variant with truncated copper-binding α helices and bilirubin oxidase free of Met-rich domains also showed such reductive inactivation process, which suggests that multicopper oxidases contain copper-binding sites that lead to inactivation.

Introduction

Multicopper oxidases (MCOs) are essential enzymes in many organisms and have been widely studied in the biochemistry, electrochemistry, and spectroscopy fields [1,2]. In solution, substrates such as phenols, bilirubin, and ascorbate are oxidized at type I (T1) Cu, and the extracted electrons are transferred to the trinuclear copper cluster (TNC) composed of one type II (T2) Cu and two type III (T3) Cu moieties, where dioxygen (O₂) is reduced into water [1,2]. MCOs are often utilized as O₂-reducing cathodic catalysts for bioelectrochemical applications, such as O₂ biosensors and biofuel cells [3,4]. They can undergo enzymatic reactions on electrode materials that act as electron donors and react with the T1 Cu. Such direct electrical communication between an enzyme and an electrode is called direct electron transfer (DET)-type bioelectrocatalysis [5], [6], [7], [8], [9]. For such purposes, carbon nanotube (CNT) networks are widely used as efficient platforms for DET-type bioelectrocatalysis of various enzymes including MCOs [10], [11], [12], [13].

Copper efflux oxidase (CueO) belongs to the MCO family. It is supposed to be able to protect periplasmic enzymes from copper mediated toxicity by oxidizing the harmful cuprous ion (Cu⁺) [14], [15], [16], [17]. Consequently, CueO is proposed to play an important role as a radical scavenger in bacterial copper homeostasis, although the exact mechanism is largely unknown. Escherichia coli (E. coli) CueO has been the most studied among CueO-type enzymes. Unlike other MCOs such as laccase (Lac) or bilirubin oxidase (BOD), the uniqueness of the E. coli CueO structure is associated with a large segment composed of α helices (helices 5, 6, and 7 from the N-terminus) that cover the T1 Cu site, which results in low catalytic activity toward the oxidation of large electron-donating substrates such as 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) [18,19]. In contrast, the helical region provides additional copper-binding sites; consequently, ABTS-oxidizing activity is improved in the presence of excess cupric ion (Cu²⁺), because the bound coppers mediate the electron transfer between ABTS and the T1 Cu [18], [19], [20]. In bioelectrocatalysis, on the other hand, E. coli CueO exhibits strong DET-type activity on positively charged electrodes because the surface charge near the T1 Cu site is negative [21]. Hence, positively charged platforms can electrostatically control the enzyme orientation in a manner favorable for the interfacial electron transfer from the electrode to the T1 Cu [21].

Our group recently studied the bioelectrocatalytic reduction of O₂ by Lac from Thermus thermophilus (TtLac). TtLac structure shows a copper-binding Met-rich hairpin domain near the T1 Cu, thus presenting similarity with E. coli CueO [22]. As with E. coli CueO, modification of electrodes by positively charged CNTs were found to be favorable for DET, while negative ones prevented DET process. For the first time, it was however demonstrated that addition of Cu²⁺ allowed bioelectrocatalytic O₂ reduction at negative CNTs, at a potential lower than the expected potential for a catalytic process passing through the T1 Cu, hence suggesting a change in the electron transfer pathway between the enzyme and the electrode. The process was tentatively attributed to the cuprous oxidase activity of the enzyme induced by Cu binding to the Met-rich domain. On positively charged CNTs where DET was favored, Cu²⁺ addition induced progressive DET current decrease with simultaneous occurrence of the Cu²⁺-related catalytic wave. Whatever positive or negative CNTs-based electrodes, voltammograms recorded in the presence of Cu²⁺ were peak-shaped. While the cause of this observation remained unknown, it was suggested that Cu²⁺-related electrocatalytic activation may be accompanied by an inactivation process. MCO inhibition by H₂O₂ and halides have been reported [23], [24], [25], [26], [27], [28]. As far as we know, MCO inactivation by Cu²⁺ was never reported.

In this study, with the final objective of improving the understanding of copper homeostasis, we examined how Cu²⁺ affects the bioelectrocatalytic properties of CueO, with a special focus on the inactivation caused by Cu²⁺. We especially analyzed kinetic data in order to discuss a potential inhibition mechanism. In addition, we investigated how the helical structure affects the bioelectrocatalytic properties of wild-type CueO by comparing it with its variant lacking Met-rich α helices and another MCO lacking any Met-rich domains.