Active-site tuning in PrnB: Resonance Raman evidence for substrate-assisted proton delivery and compound 0 reactivity

PrnB catalyzes a chemical transformation distinct from those of its homologs, tryptophan and indoleamine dioxygenases (TDO and IDO), highlighting the need for detailed characterization of its active site to elucidate the basis of its unique mechanism. Resonance Raman (rR) spectroscopy was used to probe PcPrnB from Pseudomonas chlororaphis in substrate-free and 7-Cl-L-Trp-bound forms in ferric, ferrous, and ferrous CO states. The studies revealed that, in the ferric state, the substrate directly coordinates to the heme iron, inducing conversion to a fully six-coordinate, low-spin configuration. This process is accompanied by increased heme planarity and reorganization of propionate hydrogen-bonding interactions, resulting in a more coplanar orientation of the heme vinyl substituents, which can increase their electronwithdrawing character and alter the heme reduction potential. Interestingly, the strength of the functionally important Fe–N His bond is comparable to that of TDO and IDO enzymes, implying that the proximal environment does not underlie the distinct reactivity of PrnB. Analysis of ferrous CO adducts reveals that substrate binding displaces distal water and enables hydrogen bonding between the substrate amine and the bound exogenous ligands. pH-dependent studies provide strong evidence that the substrate amine functions as a key hydrogen-bond and proton donor to both CO and physiologically relevant oxy species. These findings point to a substrate-assisted mechanism in which the substrate actively modulates the distal pocket environment and facilitates proton delivery. Together with prior work, the results support Compound 0 as the reactive intermediate in PrnB catalysis.