A DI-HEME CYTOCHROME-C PEROXIDASE FROM NITROSOMONAS-EUROPAEA CATALYTICALLY ACTIVE IN BOTH THE OXIDIZED AND HALF-REDUCED STATES

A di c-heme containing cytochrome (cytochrome c(553) peroxidase) has b een isolated from the chemoautotrophic bacterium Nitrosomonas europaea . Sequence analysis of the N terminus and the two heme-containing pept ides generated by digestion of the enzyme with trypsin show 40% homolo gy overall to sequences reported for the di-heme peroxidase from Pseud omonas aeruginosa (Ronnberg, M., Kalkkinen, N., and Ellfolk, N. (1989) FEBS Left. 250, 175-178). At room temperature and pH 7.0, one heme is low spin with E(m7) = +450 mV and the other is high spin with E(m7) = -260 mV. EPR spectra show a mixture of high spin and low spin signals at cryogenic temperatures. Anionic ligands (CN-, N-3(-), F-, CNO-) bi nd so as to perturb the high spin heme when cytochrome c(553) peroxida se is either fully oxidized (Fe-LS(3+):Fe-HS(3+)) or half-reduced (Fe- LS(2+):Fe-HS(3+)). The EPR signal of the high potential, low spin heme in fully oxidized enzyme is unperturbed by the presence of the ligand s. Furthermore, each ligand results in similar characteristic EPR sign als for either oxidation state of the peroxidase. Both the fully oxidi zed and half-reduced oxidation states of cytochrome c(553) perocatalyt ically active as evidenced by the enzyme's ability to oxidize horse he art cytochrome c in the presence of H2O2, as well as by optical change s associated with the addition of H2O2 to the peroxidase. In the prese nce of stoichiometric amounts of H2O2, the half-reduced enzyme is rapi dly oxidized and the fully oxidized enzyme shows a significant decreas e in absorbance in the Soret region of the optical spectrum coupled wi th a lesser increase near 600-650 nm. These latter optical changes are similar to what is observed in the formation of a porphyrin cation ra dical. This suggests that this di-heme peroxidase may form a compound I intermediate analogous to that formed by horseradish peroxidase.