Spectroscopic characterization and co-crystal structure of P450Blt reveal the molecular basis for biarylitide formation.

Abstract

P450Blt is a cytochrome P450 enzyme (CYP) that forms crosslinked biarylitides by installing an intramolecular aromatic CN bond on a ribosomally synthesized pentapeptide. Closely related CYPs, such as the nitrating enzyme RufO, exhibit distinct catalytic selectivity on the same peptide scaffold. Although both P450Blt and RufO have been characterized, direct mechanistic comparison has been limited by the lack of spectroscopic studies of P450Blt and differences in their crystallization methods. Here, we performed spectroscopic and structural characterization of P450Blt to establish a framework for understanding this emerging family of pentapeptide-modifying CYPs. P450Blt efficiently catalyzed Tyr-3 and His-5 coupling of its peptide substrate using heterologous ferredoxin-dependent redox systems and remained active through the peroxide shunt pathway. Spectroscopies showed that peptide binding minimally perturbs the heme structure, closely resembling RufO. Transient kinetics identified a ferric superoxo intermediate that formed more slowly than in RufO and was markedly stabilized in the presence of the peptide. A 1.81 Å co-crystal structure of the binary complex complements the previously reported peptide-soaked structure by capturing a more catalytically relevant substrate-bound state, revealing a low-spin heme center, peptide side-chain movements, and peptide-induced active site rearrangements. Comparison with the co-crystal structure of RufO demonstrates that substrate recognition and the Tyr-3 binding site are highly conserved, whereas the positioning of His-5 and its associated hydrogen-bonding network remain the principal structural differences between the two enzymes. These findings suggest that subtle active site differences modulate substrate positioning, oxygen activation, and likely proton transfer for selective catalysis.