A Distinctive Electrocatalytic Response from the Cytochrome c Peroxidase of Nitrosomonas europaea

Abstract

Here the cytochrome c peroxidase (CcP) from Nitrosomonas europaea is examined using the technique of catalytic protein film voltammetry. Submonolayers of the bacterial diheme enzyme at a pyrolytic graphite edge electrode give catalytic, reductive signals in the presence of the substrate hydrogen peroxide. The resulting waveshapes indicate that CcP is bound non-covalently in a highly active configuration. The native enzyme has been shown to possess two heme groups of low and high potential (L and H, ؊260 and ؉450 mV versus hydrogen, respectively), and here we find that the catalytic waves of the N. europaea enzyme have a midpoint potential of >500 mV and a shape that corresponds to a 1-electron process. The signals increase in magnitude with hydrogen peroxide concentration, revealing Michaelis-Menten kinetics and K m ‫؍‬ 55 M. The midpoint potentials shift with substrate concentration, indicating the electrochemically active species observed in our data corresponds to a catalytic species. The potentials also shift with respect to pH, and the pH dependence is interpreted in terms of a two pK a model for proton binding. Together the data show that the electrochemistry of the N. europaea cytochrome c peroxidase is unlike other peroxidases studied to date, including other bacterial enzymes. This is discussed in terms of a catalytic model for the N. europaea enzyme and compared with other cytochrome c peroxidases.

Direct Electrochemistry of Redox Enzymes as a Tool for Mechanistic Studies

“…331,332 As noted by Andreu and co-workers, 163 in most of these studies, catalysis occurs at an electrode potential (typically <0.1 V Vs SHE at pH 7) close to the reduction potential of the Fe III/II couple of the active site; but this redox couple is not part of the normal catalytic cycle which involves the high-potential two-electron transformation between a ferryl iron close to a radical cation (the so-called "Compound I") and a ferric state. In contrast, catalytic reduction of H 2 O 2 was observed at potentials more consistent with the involvement of Compound I for ccp, 175,285,333,334 a bacterial (diheme) ccp (Figure 27), 250,330 and horseradish peroxidase. 163 Similarly for laccases, oxygen reduction is expected to occur at very high electrode potential ( Figure 25); 77,203,211,335 otherwise, the electrochemical signal may not divulge much about the physiological catalytic process.…”

“…The low potential Fe P III/II couple is not involved in the catalytic cycle. In a recent electrochemical study of the peroxidase from Nitrosomonas europaea, 330,250 Elliott and co-workers emphasized important differences between this enzyme and other di-heme peroxidases. First, the enzyme does not display any evidence of redox-linked activation, as required in other di-heme peroxidases.…”

Protonation and inhibition of Nitrosomonas europaea cytochrome c peroxidase observed with protein film voltammetry

“…We have recently utilized direct electrochemistry as a means to investigate the redox chemistry and catalytic mechanism of Ne CCP, as an example of those CCP enzymes which do not require pre-reduction for maximal activity [15]. Regardless of the overall similarity between Ne CCP and those enzymes that do require pre-reduction, the Nitrosomonas enzyme displayed highly reversible catalytic voltammetry, marked by a high potential (>500 mV vs. NHE).…”

Mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by P. pantotrophus pseudoazurin: kinetics of intermolecular electron transfer

“…Few works dealing with the electrochemistry of other peroxidases have been published, with monohaem CCP being the most studied [24,25]. The first reports on bacterial CCPs, from P. pantotrophus and from Nitrosomonas europaea, were recently published [26,27].…”