MOSSBAUER CHARACTERIZATION OF PARACOCCUS-DENITRIFICANS CYTOCHROME-C PEROXIDASE - FURTHER EVIDENCE FOR REDOX AND CALCIUM BINDING-INDUCED HEME-HEME INTERACTION

Mossbauer and electron paramagnetic resonance (EPR) spectroscopies wer e used to characterize the diheme cytochrome c peroxidase from Paracoc cus denitrificans (L.M.D. 52.44). The spectra of the oxidized enzyme s how two distinct spectral components characteristic of low spin ferric hemes (S = 1/2), revealing different heme environments for the two he me groups, The Paracoccus peroxidase can be non-physiologically reduce d by ascorbate, Mossbauer investigation of the ascorbate-reduced perox idase shows that only one heme (the high potential heme) is reduced an d that the reduced heme is diamagnetic (S = 0), The other heme (the lo w potential heme) remains oxidized, indicating that the enzyme is in a mixed valence, half-reduced state, The EPR spectrum of the half-reduc ed peroxidase, however, shows two low spin ferric species with g(max) = 2.89 (species I) and g(max) = 2.78 (species II). This EPR observatio n, together with the Mossbauer result, suggests that both species are arising from the low potential heme, More interestingly, the spectrosc opic properties of these two species are distinct from that of the low potential heme in the oxidized enzyme, providing evidence for heme-he me interaction induced by the reduction of the high potential heme, Ad dition of calcium ions to the half-reduced enzyme converts species II to species I. Since calcium has been found to promote peroxidase activ ity, species I may represent the active form of the peroxidatic heme.