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

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 -171mV 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 83s-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 (+140mV or -195mV). Apparent Michaelis constants KM app 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. © 2012 Elsevier B.V.