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 [FeIVO] 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 [FeO] 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 [FeO] species for all three substrates, identifying the intermediates along the O2 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 [FeO] structure, in which the oxido is directed away from the substrate.

When investigating the mode of hydrogen activation by [Fe] hydrogenases, not only is the chemical reactivity at the active site of importance but also the large-scale conformational change between the so-called open and closed conformations, which leads to a special spatial arrangement of substrate and iron cofactor. To study H2 activation, a complete model of the solvated and cofactor-bound enzyme in complex with the substrate methenyl-H4MPT+ was constructed. Both the closed and open conformations were simulated with classical molecular dynamics on the 100 ns time scale. Quantum-mechanics/molecular-mechanics (QM/MM) calculations on snapshots then revealed the features of the active site that enable the facile H2 cleavage. The hydroxyl group of the pyridinol ligand can easily be deprotonated. With the deprotonated hydroxyl group and the structural arrangement in the closed conformation, H2 coordinated to the Fe center is subject to an ionic and orbital push–pull effect and can be rapidly cleaved with a concerted hydride transfer to methenyl-H4MPT+. An intermediary hydride species is not formed.

When investigating the mode of hydrogen activation by [Fe] hydrogenases, not only is the chemical reactivity at the active site of importance but also the large-scale conformational change between the so-called open and closed conformations, which leads to a special spatial arrangement of substrate and iron cofactor. To study H2 activation, a complete model of the solvated and cofactor-bound enzyme in complex with the substrate methenyl-H4MPT+ was constructed. Both the closed and open conformations were simulated with classical molecular dynamics on the 100 ns time scale. Quantum-mechanics/molecular-mechanics (QM/MM) calculations on snapshots then revealed the features of the active site that enable the facile H2 cleavage. The hydroxyl group of the pyridinol ligand can easily be deprotonated. With the deprotonated hydroxyl group and the structural arrangement in the closed conformation, H2 coordinated to the Fe center is subject to an ionic and orbital push–pull effect and can be rapidly cleaved with a concerted hydride transfer to methenyl-H4MPT+. An intermediary hydride species is not formed.