Artefacts induced on c-type haem proteins by electrode surfaces.
In this work it is demonstrated that the characterization of c-type haem containing proteins by electrochemical techniques needs to be cautiously performed when using pyrolytic graphite electrodes. An altered form of the cytochromes, which has a redox potential 300 mV lower than that of the native state and displays peroxidatic activity, can be induced by interaction with the pyrolytic graphite electrode. Proper control experiments need to be performed, as altered conformations of the enzymes containing c-type haems can show activity towards the enzyme substrate. The work was focused on the study of the activation mechanism and catalytic activity of cytochrome c peroxidase from Paracoccus pantotrophus. The results could only be interpreted with the assignment of the observed non-turnover and catalytic signals to a non-native conformation state of the electron-transferring haem. The same phenomenon was detected for Met-His monohaem cytochromes (mitochondrial cytochrome c and Desulfovibrio vulgaris cytochrome c-553), as well as for the bis-His multihaem cytochrome c(3) from Desulfovibrio gigas, showing that this effect is independent of the axial coordination of the c-type haem protein. Thus, the interpretation of electrochemical signals of c-type (multi)haem proteins at pyrolytic graphite electrodes must be carefully performed, to avoid misassignment of the signals and incorrect interpretation of catalytic intermediates.
Requimte, Centro de Química Fina e Biotecnologia, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516, Caparica, Portugal. patricia.sousa@dq.fct.unl.pt
Patrícia M
PM
Paes de Sousa
Sofia R
SR
Pauleta
M Lurdes
ML
Simões Gonçalves
Graham W
GW
Pettigrew
Isabel
I
Moura
José J G
JJ
Moura
Margarida M
MM
Correia dos Santos
D002244:Carbon / Q000737:chemistry*; D003578:Cytochrome-c Peroxidase / Q000737:chemistry; D004563:Electrochemistry / Q000379:methods*; D004566:Electrodes; D006420:Hemeproteins / Q000737:chemistry*; D042084:Paracoccus pantotrophus / Q000201:enzymology; D017433:Protein Structure, Secondary
D006420:Hemeproteins; C009139:pyrolytic carbon; D002244:Carbon; D003578:Cytochrome-c Peroxidase