PH-DEPENDENCE AND STRUCTURAL INTERPRETATION OF THE REACTIONS OF COPRINUS-CINEREUS PEROXIDASE WITH HYDROGEN-PEROXIDE, FERULIC ACID, AND 2,2'-AZINOBIS(3-ETHYLBENZTHIAZOLINE-6-SULFONIC ACID)

Steady-state and transient-state analysis of Coprinus cinereus peroxid ase, CIP (identical to Arthromyces ramosus peroxidase), was used to ch aracterize the kinetics of the three fundamental steps in heme peroxid ase catalysis: compound I(cpd I) formation, cpd I reduction, and compo und II (cpd II) reduction. The rate constant k(1) for cpd I formation determined by transient-state analysis is (9.9 +/- 0.6) x 10(6) M-1 s( -1). The k(1) determined by steady-state analysis is (8.8 +/- 0.6) x 1 0(6) M-1 s(-1) in the presence of ferulic acid and (6.7 +/- 0.2) x 10( 6) M-1 s(-1) in the presence of ABTS. The value of k(1) is constant fr om pH 6 to 11. However, at low pH the value of k(1) decreases, corresp onding to titration of an enzyme group with a pK(a) of 5.0, Titration of this group is also detected from cyanide-binding kinetics, Furtherm ore, titration of this group is linked with marked spectroscopic chang es unique to CIP, We ascribe these changes to protonation of proximal His183, A very low pK(a) is proposed for distal His55 in the resting s tate of CIP. The rate constants, k(2) for cpd I and k(3) for cpd II re duction, are very large for both ferulic acid and 2,2'-azinobis(3-ethy lbenzthiazoline-6-sulfonic acid) (ABTS), For ferulic acid, transient-s tate kinetic analysis shows that the values of k(2) and k(3) are ident ical at pH 5-6, and the ratio k(2)/k(3) increases to 10 at pH 10. The similar magnitude of k(2) and k(3) is unusual for a peroxidase. Both k (2) and k(3) decrease with increasing pH, and both are influenced by t wo ionizations: one with a pK(a) value near 7, assumed to reflect the protonation of His55; and the other with pK(a) of 9.0 +/- 0.7 for k(2) and 8.8 +/- 0.4 for k(3), perhaps reflecting the phenol-linked deprot onation of ferulic acid. Steady-state analysis at pH 7.0 gave k(2)k(3) /( k(2) + k(3)) = (2.2 +/- 0.1) x 10(7) M-1 s(-1) for ferulic acid, an d (2.0 +/- 0.7) x 10(7) M-1 s(-1) for ABTS and revealed a unimolecular step with k(n) = 1500 s(-1), ascribed to slow ABTS radical product re lease. From transient-state results at pH 7, the values of k(2), and k (3) were found to be identical also for ABTS. A mechanism for cpd I an d II reduction involving distal histidine and arginine is proposed.