Direct electrochemistry and Os-polymer-mediated bioelectrocatalysis of NADH oxidation by Escherichia coli flavohemoglobin at graphiteelectrodes

Abstract

a b s t r a c tEscherichia coli flavohemoglobin (HMP), which contains one heme and one FAD as prosthetic groups and is capable of reducing O 2 by its heme at the expense of NADH oxidized at its FAD site, was electrochemically studied at graphite (Gr) electrodes. Two signals were observed in voltammograms of HMP adsorbed on Gr, at À 477 and À 171 mV vs. Ag9AgCl, at pH 7.4, correlating with electrochemical responses from the FAD and heme domains, respectively. The electron transfer rate constant for ET reaction between FAD of HMP and the electrode was estimated to be 83 s. Direct bioelectrocatalytic oxidation of NADH by HMP was not observed, presumably due to impeded substrate access to HMP orientated on Gr through the FAD-domain and/or partial denaturation of HMP. Bioelectrocatalysis was achieved when HMP was wired to Gr by the Os redox polymers, with the onset of NADH oxidation at the formal potential of the particular Os complex (þ140 mV or À 195 mV). Apparent Michaelis constants K M app and j max were determined, showing bioelectrocatalytic efficiency of NADH oxidation by HMP exceeding the one earlier shown with diaphorase, which makes HMP very attractive as a component of bioanalytical and bioenergetic devices.

Electrocatalysis of Water Oxidation by H2O‐Capped Iridium‐Oxide Nanoparticles Electrodeposited on Spectroscopic Graphite

“…22–24, 28 The η of the WOR of 0.21 V may be also considered as one of the lowest hitherto reported, being only slightly higher than that of 0.20 V reported by Zhao et al (obtained with IrO x ⋅ n H 2 O‐NP‐modified GC rotating‐disk electrode). Also, IrO x ‐NP‐modified Gr exhibited electrocatalytic current densities that were several times higher (four–fivefold, depending on pH) than those shown with IrO x ‐NP‐modified GC RDE, which may be connected both with the much higher surface area of the Gr electrodes used here, a roughness factor exceeding 10,29 and the hydrophilic character of the graphite surface, favoring interactions with the hydrophilic surface of IrO x NPs. When catalytic current densities are referred to the surface concentration of Ir V (Table 1), IrO x ‐NP Gr electrodes also demonstrate the highest specific catalytic activity, comparable only with NPs adsorbed on GC at a sub nmol cm −2 level22 (ca.…”

Implications of FAD Electrode Reaction Kinetics for Electrocatalysis of NADH Oxidation and Development of NAD‐Dependent Enzyme Electrodes

“…In other reports (not cited here) the overpotential of the reaction was still too high (over 0.6–0.8 V) and NADH oxidation might be rather related to the electrocatalytic activity of the electrode itself than to the FAD electrochemistry, e.g. to the presence of surface quinone functionalities 10e. The apparent inconsistency between the expected catalytic activity of FAD and the lack of its experimental demonstration triggered our research aimed at the development of the catalytically active FAD‐modified electrodes and their application in NAD‐dependent enzyme biosensors.…”

“…In the presence of NADH a distinct electrocatalytic current (absent at the PEI‐modified and bare Gr electrodes) can be followed starting from potentials of the FAD transformation (Figure 5). It is worth to mention that NADH oxidation at bare Gr electrodes starts at potentials higher than 50 mV, correlating with the catalysis by surface quinoid functionalities 10e (those are not detectable neither at FAD‐, PEI‐ or FAD/PEI‐modified Gr electrodes). At the PEI‐modified Gr electrodes, no catalytic oxidation of NADH can be followed within the potentials window represented in Figure 5, and at potentials over 0.4 V oxidation of PEI layer interfered with the NADH oxidation studies.…”

“…The surface concentration of FAD on Gr estimated by integrating the peak area ( Γ FAD = Q / nFA ) and referred to the geometric surface area of the electrodes ( A= 0.07 cm 2 ) was 17.7±0.5 nmol cm −2 , which is far above the monolayer coverage, even if one takes into account a high surface roughness (>10) 10e, 20 of the used Gr electrodes. For redox peak separations higher than 200/ n mV (fast potential scan rates) a value of 1.8±0.3 s −1 for the ET rate constant k s was determined by extrapolation of the linear part of the two branches (anodic and cathodic) of the E peak −log ν dependence (Figure 2B), using the following equations: …”

Development of a Photoelectrochemical Lactic Dehydrogenase Biosensor Using Multi-Wall Carbon Nanotube -Tio 2 Nanoparticle Composite as Coenzyme Regeneration Tool

“…For example, the electrochemical oxidation of the reduced form of nicotinamide adenine dinucleotide (NADH) to NAD + commonly occurs at high potentials, which may cause the interferences from other electroactive compounds usually present in real samples [19,20]. In addition, the oxidation of NADH at high potential may also damage the functionality and stability of the dehydrogenase biosensors [21].…”