Structural Characterization of Cytochrome from an Ammonia-Oxidizing Bacterium

The ammonia-oxidizing bacterium Nitrosomonas europaea expresses two cytochromes in the P460 superfamily that are predicted to be structurally similar. In one, cytochrome (cyt) P460, the substrate hydroxylamine (NH₂OH) is converted to nitric oxide (NO) and nitrous oxide (N₂O) requiring a unique heme-lysyl cross-link in the catalytic cofactor. In the second, cyt $$, the cross-link is absent, and the cytochrome instead binds H₂O₂ forming a ferryl species similar to compound II of peroxidases. Here, we report the 1.80 Å crystal structure of cyt $$─a well-expressed protein in N. europaea with a lysine to a methionine replacement at the cross-linking position. The structure of cyt $$ is characterized by a large β-sheet typical of P460 members; however, several localized structural differences render cyt $$ distinct. This includes a large lasso-like loop at the "top" of the cytochrome that is not observed in other structurally characterized members. Active site variation is also observed, especially in comparison to its closest homologue cyt $$ from the methane-oxidizing Methylococcus capsulatus Bath, which also lacks the cross-link. The phenylalanine "cap" which is presumed to control small ligand access to the distal heme iron is replaced with an arginine, reminiscent of the strictly conserved distal arginine in peroxidases and to the NH₂OH-oxidizing cytochromes P460. A critical proton-transferring glutamate residue required for NH₂OH oxidation is nevertheless missing in the active site. This in part explains the inability of cyt $$ to oxidize NH₂OH. Our structure also rationalizes the absence of a methionyl cross-link, although the side chain's spatial position in the structure does not eliminate the possibility that it could form under certain conditions.