This study provides a structural characterization of the biaryltilide cross-linking P450 enzyme, P450bit, in complex with its pentapeptide substrate MRYLH. The structure, determined at 1.8 Å resolution, reveals how the peptide is accommodated within the enzyme's active site, with key I-helix residues playing a crucial role in coordinating the histidine residue of the substrate. This coordination is central to the enzyme's specificity for generating a C–N bond between tyrosine and histidine residues in the MRYLH substrate. The structure also highlights the importance of Arg residues in coordinating the C-terminus of the peptide, which is essential for controlling the type of cross-linking outcome. Computational studies suggest that the formation of a C–N bond is energetically favorable, despite the enzyme's tendency to form a C–C bond. The analysis of related P450 enzymes and sequence comparisons further elucidate the mechanisms and specificity of these enzymes in cross-linking reactions. The findings provide valuable insights into the structural basis of peptide cross-linking by P450s and the factors that control the type of cross-linking outcome.This study provides a structural characterization of the biaryltilide cross-linking P450 enzyme, P450bit, in complex with its pentapeptide substrate MRYLH. The structure, determined at 1.8 Å resolution, reveals how the peptide is accommodated within the enzyme's active site, with key I-helix residues playing a crucial role in coordinating the histidine residue of the substrate. This coordination is central to the enzyme's specificity for generating a C–N bond between tyrosine and histidine residues in the MRYLH substrate. The structure also highlights the importance of Arg residues in coordinating the C-terminus of the peptide, which is essential for controlling the type of cross-linking outcome. Computational studies suggest that the formation of a C–N bond is energetically favorable, despite the enzyme's tendency to form a C–C bond. The analysis of related P450 enzymes and sequence comparisons further elucidate the mechanisms and specificity of these enzymes in cross-linking reactions. The findings provide valuable insights into the structural basis of peptide cross-linking by P450s and the factors that control the type of cross-linking outcome.