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

Escherichia coli flavohemoglobin (HMP), which contains one heme and one FAD as prosthetic groups and is capable of reducing O2 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 −477 and −171 mV vs. Ag|AgCl, 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−1. 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 (+140 mV or −195 mV). Apparent Michaelis constants KMapp and jmax 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.

► New NADH-oxidising enzyme Escherichia coli flavohemoglobin was electrochemically studied.
► FAD and heme redox centers of flavohemoglobin were electrochemically determined.
► Flavohemoglobin entrapped in the Os-polymer matrix bioelectrocatalytically oxidized NADH.
► Efficiency of NADH oxidation was superior to show with commonly used diaphorase.
► Results are important for the development of NAD/NADH-dependent biosensors, biofuel cells and bioelectronic devices.