R. Gilmour et al., SPECTROSCOPIC CHARACTERIZATION OF CYTOCHROME-C PEROXIDASE FROM PARACOCCUS-DENITRIFICANS, Biochemical journal, 294, 1993, pp. 745-752
The cytochrome c peroxidase of Paracoccus denitrificans is similar to
the well-studied enzyme from Pseudomonas aeruginosa. Like the Pseudomo
nas 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-potent
ial haem acts as a peroxidatic centre. Reduction with ascorbate of the
high-potential haem of the Paracoccus enzyme results in a switch of t
he 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 K(D) of 5 muM. The Paracocc
us enzyme is significantly different from that from Pseudomonas in the
time course of high-spin formation after reduction of the high-potent
ial 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 se
parated in time and the intermediate for-m obtained. This separation i
s 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 m
ixed-valence enzyme. The spin-state switch of the low-potential haem s
hifts the midpoint redox potential of the high-potential haem by 50 mV
, a further indication of haem-haem interaction.