Formation and structure of the ferryl [FeO] intermediate in the non-haem iron halogenase SyrB2: classical and QM/MM modelling agree

Abstract

To rationalise mechanistically the intriguing regio- and chemoselectivity patterns for different substrates of the non-haem iron/2-oxoglutarate dependent halogenase SyrB2, it is crucial to elucidate the structure of the pivotal [Fe[double bond, length as m-dash]O] intermediate. We have approached the problem by a combination of classical and QM/MM modelling. We present complete atomistic models of SyrB2 in complex with its native substrate l-threonine as well as l-α-amino butyric acid and l-norvaline (all conjugated to the pantetheine tether), constructed by molecular docking and extensive MD simulations. We evaluate five isomers of the [Fe[double bond, length as m-dash]O] intermediate in these simulations, with a view to identifying likely structures based on a simple "reaction distance" measure. Starting from models of the resting state, we then use QM/MM calculations to investigate the formation of the [Fe[double bond, length as m-dash]O] species for all three substrates, identifying the intermediates along the O activation/decarboxylation pathway on the S = 1, 2, and 3 potential-energy surfaces. We find that, despite differences in the detailed course of the reaction, essentially all pathways produce the same [Fe[double bond, length as m-dash]O] structure, in which the oxido is directed away from the substrate.

Are Vanadium Intermediates Suitable Mimics in Non-Heme Iron Enzymes? An Electronic Structure Analysis

“…Isomerization of the active site in non-heme iron halogenases is expected to play an important role in substrate positioning and reaction selectivity 32,87 . Since it is hard to capture these fleeting iron intermediates experimentally, numerous computational studies [83][84][88][89][90][91] have been carried out to investigate the mechanism of selective halogenation by non-heme iron halogenases. While the crystal structures obtained with Fe(II)/aKG suggest the formation of axial oxo 28,32,92 in most cases, isomerization to equatorial oxo is possible 40,85 .…”

The Protein’s Role in Substrate Positioning and Reactivity for Biosynthetic Enzyme Complexes: The Case of SyrB2/SyrB1

“…However, most modeling has been carried out on cluster models of SyrB2, with few exceptions 36,38 , despite the wider use of whole-protein multi-scale modeling for other non-heme iron enzymes [46][47][48][49][50] . To date, no SyrB2/SyrB1 complexes have been simulated, with the largest models using truncated 34,[36][37][38] PPant-S-Thr models placed in SyrB2 by rotating or mutating F196. Shedding insight into structural features of the SyrB2/SyrB1 complex, Silakov and coworkers 51 recently employed hyperfine sublevel correlation (HYSCORE) 52 spectroscopy to estimate average positions and angles of PPant-loaded substrate methyl and ethyl carbons with respect to the iron center in the active site of the SyrB2/SyrB1 complex, similar to previous efforts that had provided insight into non-heme iron hydroxylases [53][54] .…”

The Protein’s Role in Substrate Positioning and Reactivity for Biosynthetic Enzyme Complexes: the Case of SyrB2/SyrB1

“…Thus it appears that SyrB2 has a substrate-specific catalytic inefficiency for the native substrate that coincides with halogenation being favored over hydroxylation 32 . This unexpected behavior has sparked vigorous computational [33][34][35][36][37][38] and spectroscopic [39][40] study of SyrB2 to better understand its unexpected reactivity. One suggestion 37,[40][41] has been that configurational isomerization of the active site, especially after formation of the oxo intermediate 38,41 , reorients Cl to an axial position to make it more accessible for halogenation.…”