Kinetic analysis of manganese peroxidase. The reaction with manganese complexes

Manganese peroxidase from the lignin-degrading fungus Phanerochaete chrysosporium catalyzes the H₂O₂-dependent oxidation of Mn²⁺ to Mn³⁺. Presteady-state methods were employed to characterize the reactions of free and chelated Mn²⁺ with the 2-electron and 1-electron oxidized forms of the enzyme, compounds I and II, respectively. At pH 4.5, the optimum pH for steady-state turnover, the reaction of compound I with Mn²⁺, either free or complexed, is too rapid to measure by stopped flow methods. The reactions of compound I with Mn²⁺ can only be monitored under non-optimal conditions of pH 2.5. The reaction of compound II with Mn²⁺ is much slower than compound I. Chelators such as oxalate, lactate, and malonate facilitated the reaction of Mn²⁺ with compound II. In contrast, succinate, which does not readily form a complex with Mn²⁺, and polyglutamate, which is polymeric, were ineffective in stimulating the reaction of Mn²⁺ with compound II. The 1:1 chelator-Mn²⁺ complex is the preferred substrate for compound II; this conclusion is based on known formation constants for the various Mn²⁺ complexes. Steady-state kinetics studies were performed by directly measuring the initial rate of Mn³⁺ formation. The k(cat) values for the formation of Mn³⁺-oxalate, Mn³⁺-lactate, and Mn³⁺-malonate are 308, 211, and 220 s^{- 1}, respectively. The K(m) values for Mn²⁺-oxalate, Mn²⁺-lactate, and Mn²⁺-malonate are 13, 41, and 18 μM, respectively. These results collectively indicate that manganese peroxidase does not readily oxidize free (hexa-aquo) Mn²⁺ as previously proposed (Wariishi, H., Valli, K., and Gold, M. H. (1992) J. Biol. Chem. 267, 23688-23695), but the Mn²⁺ has to be chelated to support steady-state turnover.