Assessing electron transfer reactions and catalysis in multicopper oxidases with operando X-ray absorption spectroscopy

Abstract

Here we propose an experimental setup based on operando X-ray absorption spectroscopy (XAS) to understand why copper-containing oxidoreductase enzymes show exceptional performance as catalysts for the oxygen reduction reaction (ORR). An electrode based on carbon nanoparticles organized in mesoporous structures with bilirubin oxidase (BOD) was developed to be used in a home-made operando XAS electrochemical cell, and we probed the electron transfer under ORR regime. In the presence of molecular oxygen, the BOD cofactor containing 4 copper ions require an overpotential about 150 mV to be reduced as compared to that in the absence of oxygen. A second electron transfer step, which occurs faster than the cofactor reduction, suggests that the cooper ions act as a tridimensional redox active electronic bridges for the electron transfer reaction.

Stabilization of Bilirubin Oxidase in a Biogel Matrix for High-performance Gas Diffusion Electrodes

“…(Christenson et al, 2006;Schubert et al, 2009;Tsujimura et al, 2005b) The second redox couple with E o equal to 0.16 V is in good agreement with the data reported for reduction and oxidation of the T2/T3 Cu center of the enzyme, (Ivnitski et al, 2008) which acts as an electronic bridge in the internal electron transfer mechanism to promote the reduction of dioxygen. (Macedo et al, 2020) As previously reported, the peaks corresponding to reduction and oxidation of the T1 Cu site are less defined in neutral media as a result of the change of bond length between this Cu atom and the ligand groups and the special arrangement of this site. (Ivnitski et al, 2008;Ramírez et al, 2008) These results indicated an efficient direct electrical communication between the enzyme and the carbon nanoparticles, which is usually desirable in enzyme-based BFCs and biosensors.…”

“…The deposition of matter along the original drop edge after the evaporation of a solvent is known as the coffee-ring effect. (Mampallil and Eral, 2018) This phenomenon has been reported during the redox enzyme immobilization (Li et al, 2018) and happens during the solvent evaporation, when the edges of the drop become pinned to the solid substrate, and the outward capillary flow from the center of the drop brings the nonvolatile component to the edge of the droplet. (Crivoi and Duan, 2013) Characteristics similar to those of the BOD-based biogel and bare-BOD films immobilized on the gold substrate are also obtained for the glassy carbon (GC) platform; the BOD-based biogel film (Fig.…”

“…Initially, the Nafion® 117 solution (5%) was diluted in the phosphate buffer, pH 7.2, at a 1:1 (v/v) ratio to obtain a Nafion salt solution and to prevent denaturation of the enzyme by local acidification. (Macedo et al, 2020) The GA solution (25%) was also diluted in the phosphate buffer (pH 7.2) at a 1:10 (v/v) ratio. Then, a 0.14-mg μL -1 BOD solution in phosphate buffer (pH 7.2) was mixed with the Nafion diluted salt solution and GA diluted solution at a 1:1:1 (v/v/v) ratio.…”

From Enzyme Stability to Enzymatic Bioelectrode Stabilization Processes

“…Methods allowing mass or layer thickness quantification, i.e., SPR, QCM-D and ellipsometry, coupled to electrochemistry will give access to the relationship between enzyme loading or release and catalytic response [21,116,271,272,311]. Electrochemistry coupled to spectroscopies (IR (including SEIRA, PMIRRAS), XAS, fluorescence, Raman (including SERRS), EPR, CD, mass spectrometry) will help in the determination of conformational change once enzyme is immobilized or as a function of applied potential or current [312][313][314][315][316][317]. Microscopy coupled to electrochemistry will allow the mapping of the electrocatalysis [318][319][320].…”

In Situ and Operando Techniques for Investigating Electron Transfer in Biological Systems

“…Within the vast number of spectroscopies, some are more commonly used for in situ and operando measurements, such as Fourier transform infrared (FTIR) spectroscopy that provides information on the vibrational modes of covalent bonds within a given molecule, [4] while X‐ray absorption spectroscopy (XAS) provides information on the oxidation state of redox co‐factors and their chemical environment/coordination sphere [5] . Raman spectroscopy, another powerful vibrational spectroscopic technique, is widely used for the chemical and structural characterization of biomolecules and studies the inelastic interaction of molecules with monochromatic light, usually emitted by a laser source in the visible, near‐IR, or near‐ultraviolet (UV) range [6] .…”