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

Abstract The impact of protonation and inhibitor binding of the diheme cytochrome c peroxidase (CCP) from Nitrosomonas europaea has been examined by the technique of catalytic protein film voltammetry (PFV). Previous efforts have shown that the low-potential heme active site ( L ) binds substrate and yields electrocatalysis at an pyrolytic graphite edge electrode, with properties evocative of a high-potential intermediate, with E m  > 540 mV (vs. normal hydrogen electrode) [A.L. Bradley, S.E. Chobot, D.M. Arciero, A.B. Hooper, S. J. Elliott, J. Biol. Chem. 279 (2004) 13297–13300]. Here we demonstrate through similar experiments that catalytic PFV generates limiting currents which allow for electrochemically-detected enzymology of the Ne CCP: such as the demonstration that pH-dependent Michaelis–Menten constants ( K m values) reveal a pK a value of 6.5 associated with the “ES” complex. Further, the direct electrocatalysis is shown in the presence of known inhibitors (cyanide and azide), indicating that inhibitor binding occurs at L , and shifts the resulting catalytic midpoint potential in a negative direction. Michaelis–Menten treatment of the limiting currents generated in the presence of variable concentrations of inhibitors showed that cyanide behaved as a competitive inhibitor with a K i value of 0.15 μM; azide revealed a mixed-mode of inhibition. The observed data were found to support a previous model of electrocatalysis, and the role of proton transfer chemistry in the active site is discussed in terms of a structural model.