Mechanism of Peroxide-Dependent Heme-Lysine Cross-Link Formation in Cytochrome P460 from N. europaea : Cpd I-Triggered meso -Carbon Hydroxylation and C–N Bond Formation

In heme c, protoporphyrin IX employs its vinyl groups to form covalent thioether bonds with the side chains of cysteine residues. The special "heme P460" cofactors in heme c include the additional heme-lysine cross-link. Previous studies have confirmed that the peroxide can drive the formation of a heme-lysine cross-link. Here, we explored the formation mechanism of the heme-Lys cross-link by performing QM/MM calculations. Our calculation results revealed that the Fe(III)-coordinated H₂O₂ first undergoes a heterolytic O-O cleavage to generate Cpd I, which then employs the water molecule in the active site to carry out the meso-hydroxylation of porphyrin, followed by nucleophilic attack of lysine on the meso-carbon. Importantly, the water molecule generated during the formation of Cpd I is the key to the hydroxylation of the meso-carbon. The pocket residues and the propionic acid side chain of heme not only constitute an encirclement to prevent the escape of the water molecule but also act as a catalytic base/acid to mediate the proton transfer and dehydration processes. Two key factors are responsible for the formation of the heme-Lys cross-link in cytochrome P460s: the fixed water molecule in the reaction center and the suitable orientation of Lys70 for the nucleophilic attack on the unsaturated meso-carbon.