P450 versus P420: correlation between cyclic voltammetry and visible absorption spectroscopy of the immobilized heme domain of cytochrome P450 BM3.
Cyclic voltabsorptometry is used for the first time to distinguish and characterize electrochemically the active (P450) and inactive (P420) forms of cytochromes P450 immobilized on an electrode during voltammetry experiments. This was achieved by using the heme domain (BMP) of the bacterial cytochrome P450 BM3 from Bacillus megaterium (CYP102A1) immobilized on mesopouros tin-oxide (SnO2) electrodes. We demonstrate that the formation of either the P450 form or the P420 one can be obtained by modifying the mesoporous electrode surface with polycations with different properties such as polyethylenimmine (PEI) and polydiallyldimethylammonium chloride (PDDA). Potential step spectroelectrochemistry allowed measurement of reduction potentials of the active P450 form. Values of -0.39+/-0.01 V and -0.58+/-0.01 V (both versus Ag/AgCl) were calculated for the active P450 form immobilized on the BMP/PDDA-SnO2 and BMP/PEI-SnO2 electrodes, respectively. The cyclic voltabsorptometric experiments showed how, when both the active and inactive forms are present on the PEI film, the inactive P420 species tends to dominate the cyclic voltammetric signal.
Panicco
Paola
P
Division of Molecular Biosciences and Department of Chemistry, Imperial College London, South Kensington, London SW7 2AZ, UK.
Astuti
Yeni
Y
Fantuzzi
Andrea
A
Durrant
James R
JR
Gilardi
Gianfranco
G
D001410:Bacillus megaterium / Q000201:enzymology*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D003580:Cytochromes / Q000737:chemistry* / Q000378:metabolism; D004563:Electrochemistry; D004566:Electrodes; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D007097:Imines / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry; D011095:Polyethylenes / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D013057:Spectrum Analysis; D017971:Tin Compounds / Q000737:chemistry; D017186:Titrimetry
D003580:Cytochromes; D007097:Imines; D011095:Polyethylenes; D000644:Quaternary Ammonium Compounds; D017971:Tin Compounds; C041004:poly-N,N-dimethyl-N,N-diallylammonium chloride; D006418:Heme; C013464:cytochrome P420; D003577:Cytochrome P-450 Enzyme System; D010100:Oxygen