THE KINETICS OF THE OXIDATION OF CYTOCHROME-C BY PARACOCCUS CYTOCHROME-C PEROXIDASE

In work that is complementary to our investigation of the spectroscopi c features of the cytochrome c peroxidase from Paracoccus denitrifican s [Gilmour, Goodhew, Pettigrew, Prazeres, Moura and Moura (1993) Bioch em. J. 294, 745-752], we have studied the kinetics of oxidation of cyt ochrome c by this enzyme. The enzyme, as isolated, is in the fully oxi dized form and is relatively inactive. Reduction of the high-potential haem at pH 6 with ascorbate results in partial activation of the enzy me. Full activation is achieved by addition of 1 mM CaCl2. Enzyme acti vation is associated with formation of a high-spin state at the oxidiz ed low-potential haem. EGTA treatment of the oxidized enzyme prevents activation after reduction with ascorbate, while treatment with EGTA o f the reduced, partially activated, form abolishes the activity. We co nclude that the active enzyme is a mixed-valence form with the low-pot ential haem in a high-spin state that is stabilized by Ca2+. Dilution of the enzyme results in a progressive loss of activity, the extent of which depends on the degree of dilution. Most of the activity lost up on dilution can be recovered after reconcentration. The M(r) of the en zyme on molecular-exclusion chromatography is concentration-dependent, with a shift to lower values at lower concentrations. Values of M(r) obtained are intermediate between those of a monomer (39565) and a dim er. We propose that the active form of the enzyme is a dimer which dis sociates at high dilution to give inactive monomers. From the activity of the enzyme at different dilutions, a K-D of 0.8 mu M can be calcul ated for the monomerdimer equilibrium. The cytochrome c peroxidase oxi dizes horse ferrocytochrome c with first-order kinetics, even at high ferrocytochrome c concentrations. The maximal catalytic-centre activit y ('turnover number') under the assay conditions used is 62000 min(-1) , with a half-saturating ferrocytochrome c concentration of 3.3 mu M. The corresponding values for the Paracoccus cytochrome c-550 (presumed to be the physiological substrate) are 85000 min(-1) and 13 mu M. How ever, in this case, the kinetics deviate from first-order progress cur ves at all ferrocytochrome c concentrations. Consideration of the peri plasmic environment in Paracoccus denitrificans leads us to propose th at the enzyme will be present as the fully active dimer supplied with saturating ferrocytochrome c-550.