Cytochrome ' Is a Variant in the P460 Superfamily Lacking the Heme-Lysyl Cross-Link: A Peroxidase Mimic Generating a Ferryl Intermediate.

A defining characteristic of bacterial cytochromes (cyt's) in the P460 family is an unusual cross-link connecting the heme porphyrin to the side chain of a lysyl residue in the protein backbone. Here, via proteomics of the periplasmic fraction of the ammonia-oxidizing bacterium (AOB) , we report the identification of a variant member of the P460 family that contains a methionyl residue in place of the cross-linking lysine. We formally designate this protein cytochrome "'" to distinguish it from other members bearing different residues at this position (e.g., cyt ' from the methane-oxidizing Bath). As isolated, the monoheme cyt ' is high-spin ( = /). Optical spectroscopy suggests that a cross-link is absent. Hydroxylamine, the substrate for the cross-linked cyt P460 from , did not appreciably alter the optical spectrum of cyt ' with up to 1000-fold excess at pH 7.5. Cyt ' did however bind 1 equiv of HO, and with a slight excess, Mössbauer spectroscopy indicated the formation of a semistable ferryl (Fe═O) Compound II-like species. The corresponding electron paramagnetic resonance showed a very low intensity signal indicative of a radical at = 2.0. Furthermore, cyt ' exhibited guaiacol-dependent peroxidase activity ( = 20.0 ± 1.2 s; = 2.6 ± 0.4 mM). Unlike cyt ', cyt P460 showed evidence of heme inactivation in the presence of 2 equiv of HO with no appreciable guaiacol-dependent peroxidase activity. Mutagenesis of the cross-linking lysyl residue to an alanine in cyt P460, however, reversed this lack of activity.