Spectroscopic characterization of cytochrome c peroxidase from Paracoccus denitrificans.
The cytochrome c peroxidase of Paracoccus denitrificans is similar to the well-studied enzyme from Pseudomonas aeruginosa. Like the Pseudomonas enzyme, the Paracoccus peroxidase contains two haem c groups, one high potential and one low potential. The high-potential haem acts as a source of the second electron for H2O2 reduction, and the low-potential haem acts as a peroxidatic centre. Reduction with ascorbate of the high-potential haem of the Paracoccus enzyme results in a switch of the low-potential haem to a high-spin state, as shown by visible and n.m.r. spectroscopy. This high-spin haem of the mixed-valence enzyme is accessible to ligands and binds CN- with a KD of 5 microM. The Paracoccus enzyme is significantly different from that from Pseudomonas in the time course of high-spin formation after reduction of the high-potential haem, and in the requirement for bivalent cations. Reduction with 1 mM ascorbate at pH 6 is complete within 2 min, and this is followed by a slow appearance of the high-spin state with a half-time of 10 min. Thus the process of reduction and spin state change can be easily separated in time and the intermediate form obtained. This separation is also evident in e.p.r. spectra, although the slow change involves an alteration in the low-spin ligation at this temperature rather than a change in spin state. The separation is even more striking at pH 7.5, where no high-spin form is obtained until 1 mM Ca2+ is added to the mixed-valence enzyme. The spin-state switch of the low-potential haem shifts the midpoint redox potential of the high-potential haem by 50 mV, a further indication of haem-haem interaction.
Department of Preclinical Veterinary Sciences, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Summerhall, Scotland, U.K.
R
R
Gilmour
C F
CF
Goodhew
G W
GW
Pettigrew
S
S
Prazeres
I
I
Moura
J J
JJ
Moura
D000596:Amino Acids / Q000032:analysis; D003486:Cyanides / Q000737:chemistry; D003578:Cytochrome-c Peroxidase / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000032:analysis; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D010231:Paracoccus denitrificans / Q000201:enzymology*; D013057:Spectrum Analysis
D000596:Amino Acids; D003486:Cyanides; D006418:Heme; D003578:Cytochrome-c Peroxidase