Showcasing two C-H bond-cleaving manifolds through mechanistic studies of isopenicillin N synthase

Abstract

The enzyme isopenicillin N synthase catalyzes the four-electron oxidation of the linear tripeptide, L-δ-aminoadipoyl- L-Cys-D-Val, to the bicyclic compound isopenicillin N, an important precursor in the biosyntheses of many β-lactam antibiotics. This reaction is catalyzed at a mononuclear non-heme-iron active site using molecular oxygen as the oxidant. Baldwin and coworkers have advanced a mechanism for this unusual reaction on the basis of extensive bioorganic and X-ray crystallographic studies, which has subsequently been supported by computational studies. The reaction proceeds in two steps. The first step forms the β-lactam ring and is initiated by cleavage of the pro-S-C_{Cys, β} -H bond. The second step forms the thiazolidine ring and begins with the cleavage of the C_Val,β-H bond. The Fe(III)-superoxo and Fe(IV)-oxo complexes are the proposed C-H-cleaving intermediates, targeting the pro-S-C_Cys,β-H and C_Val,β-H bonds, respectively. Despite significant insight into the bioorganic nature of this reaction, the bioinorganic chemistry, in particular the geometric and electronic structures of the proposed intermediates, is less well understood. In this work, we employed a combination of kinetic and spectroscopic methods, along with use of the specifically deuterated substate isotopologes, A[ d₂-C]V and AC[d₈-V], to trap and characterize both C-H-cleaving intermediates. Our results suggest that the two intermediates are best described as high-spin Fe(III)-superoxo and high-spin Fe(IV)-oxo complexes, respectively. A thorough kinetic dissection was also performed to produce a global kinetic mechanism that aligns all available data. Lastly, an attempt was made to rationalize the high selectivity of the IPNS ferryl complex to initiate thiazolidine cyclization rather than hydroxylation, a reactivity more commonly associated with ferryl intermediates.