The Heme-Lys Cross-Link in Cytochrome P460 Promotes Catalysis by Enforcing Secondary Coordination Sphere Architecture.

ABSTRACT

Cytochrome (cyt) P460 is a c-type monoheme enzyme found in ammonia-oxidizing bacteria (AOB) and methanotrophs; additionally, genes encoding it have been found in some pathogenic bacteria. Cyt P460 is defined by a unique post-translational modification to the heme macrocycle, where a lysine (Lys) residue covalently attaches to the 13' meso carbon of the porphyrin, modifying this heme macrocycle into the enzyme's eponymous P460 cofactor, similar to the cofactor found in the enzyme hydroxylamine oxidoreductase. This cross-link imbues the protein with unique spectroscopic properties, the most obvious of which is the enzyme's green color in solution. Cyt P460 from the AOB Nitrosomonas europaea is a homodimeric redox enzyme that produces nitrous oxide (N2O) from 2 equiv of hydroxylamine. Mutation of the Lys cross-link results in spectroscopic features that are more similar to those of standard cyt c' proteins and renders the enzyme catalytically incompetent for NH2OH oxidation. Recently, the necessity of a second-sphere glutamate (Glu) residue for redox catalysis was established; it plausibly serves as proton relay during the first oxidative half of the catalytic cycle. Herein, we report the first crystal structure of a cross-link deficient cyt P460. This structure shows that the positioning of the catalytically essential Glu changes by approximately 0.8 Å when compared to a cross-linked, catalytically competent cyt P460. It appears that the heme-Lys cross-link affects the relative position of the P460 cofactor with respect to the second-sphere Glu residue, therefore dictating the catalytic competency of the enzyme.