A di-heme cytochrome c peroxidase from Nitrosomonas europaea catalytically active in both the oxidized and half-reduced states.

Abstract

A di-c-heme containing cytochrome (cytochrome c553 peroxidase) has been isolated from the chemoautotrophic bacterium Nitrosomonas europaea. Sequence analysis of the N terminus and the two heme-containing peptides generated by digestion of the enzyme with trypsin show 40% homology overall to sequences reported for the di-heme peroxidase from Pseudomonas aeruginosa (Rönnberg, M., Kalkkinen, N., and Ellfolk, N. (1989) FEBS Lett. 250, 175-178). At room temperature and pH 7.0, one heme is low spin with Em7 = +450 mV and the other is high spin with Em7 = -260 mV. EPR spectra show a mixture of high spin and low spin signals at cryogenic temperatures. Anionic ligands (CN-, N3-, F-, CNO-) bind so as to perturb the high spin heme when cytochrome c553 peroxidase is either fully oxidized (FeLS3+:FeHS3+) or half-reduced (FeLS2+:FeHS3+). The EPR signal of the high potential, low spin heme in fully oxidized enzyme is unperturbed by the presence of the ligands. Furthermore, each ligand results in similar characteristic EPR signals for either oxidation state of the peroxidase. Both the fully oxidized and half-reduced oxidation states of cytochrome c553 peroxidase are catalytically active as evidenced by the enzyme's ability to oxidize horse heart cytochrome c in the presence of H2O2, as well as by optical changes associated with the addition of H2O2 to the peroxidase. In the presence of stoichiometric amounts of H2O2, the half-reduced enzyme is rapidly oxidized and the fully oxidized enzyme shows a significant decrease in absorbance in the Soret region of the optical spectrum coupled with a lesser increase near 600-650 nm. These latter optical changes are similar to what is observed in the formation of a porphyrin cation radical. This suggests that this di-heme peroxidase may form a compound I intermediate analogous to that formed by horseradish peroxidase.