Protonation and inhibition of Nitrosomonas europaea cytochrome c peroxidase observed with protein film voltammetry.
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)>540mV (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 0.15muM; 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.
Elliott
Sean J
SJ
Department of Chemistry, 590 Commonwealth Ave. Boston, MA 02215, United States. elliott@bu.edu
Bradley
Amy L
AL
Arciero
David M
DM
Hooper
Alan B
AB
D002384:Catalysis; D003578:Cytochrome-c Peroxidase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D004563:Electrochemistry; D008958:Models, Molecular; D043370:Nitrosomonas europaea / Q000201:enzymology*; D011522:Protons
D011522:Protons; D003578:Cytochrome-c Peroxidase