Electrochemical evidence for multiple peroxidatic heme states of the diheme cytochrome c peroxidase of Pseudomonas aeruginosa.
The enzyme cytochrome c peroxidase from Pseudomonas aeruginosa and its catalytic mechanism were investigated using protein film voltammetry. Monolayers of the diheme bacterial enzyme were immobilized on both pyrolytic graphite edge and alkanethiol-modified Au electrodes. The redox couple associated with the low potential heme could be detected on both electrode surfaces at a reduction potential of -234 mV vs SHE. The midpoint potential displays a distinct pH dependence at acidic pH values, indicative of proton-coupled electron transfer. The nonturnover signal of the LP heme can be transformed into sigmoidal waves upon the addition of substrate. The midpoint potentials of the turnover signals were used to calculate Michaelis-Menten kinetics with a K(m) = 25 microM. Catalysis was inhibited with addition of cyanide (K(i) = 50 microM). These kinetic parameters are in good agreement with previously reported solution-based studies, indicating that the activity of the enzyme is unaffected by the immobilization on the electrode surface. The reduction potential of the catalytic wave clearly shows that the rate-limiting species during electrocatalysis differs from those previously reported for peroxidases, indicating that PFV may be used in the future to distinguish the requirement for reductive activation in bacterial cytochrome c peroxidases.
Becker
Clinton F
CF
Department of Chemistry, Boston University, Boston, Massachusetts 02215, USA.
Watmough
Nicholas J
NJ
Elliott
Sean J
SJ
D001426:Bacterial Proteins / Q000737:chemistry*; D002384:Catalysis; D020134:Catalytic Domain; D003578:Cytochrome-c Peroxidase / Q000737:chemistry*; D004563:Electrochemistry; D004579:Electron Transport; D006418:Heme / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry
D001426:Bacterial Proteins; D008024:Ligands; D011994:Recombinant Proteins; D006418:Heme; D003578:Cytochrome-c Peroxidase