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

In heme <i>c</i>, 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 <i>c</i> 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 <i>meso</i>-hydroxylation of porphyrin, followed by nucleophilic attack of lysine on the <i>meso</i>-carbon. Importantly, the water molecule generated during the formation of Cpd I is the key to the hydroxylation of the <i>meso</i>-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 <i>meso</i>-carbon.