Prolyl-4-hydroxylase (P4H) is a non-heme iron hydroxylase that regio- and stereospecifically hydroxylates proline residues in a peptide chain into R-4-hydroxyproline, which is essential for collagen cross-linking purposes in the human body. Surprisingly, in P4H, a strong aliphatic C-H bond is activated, while thermodynamically much weaker aliphatic C-H groups, that is, at the C and C positions, are untouched. Little is known on the origins of the high regio- and stereoselectivity of P4H and many non-heme and heme enzymes in general, and insight into this matter may be relevant to Biotechnology as well as Drug Development. The active site of the protein contains two aromatic residues (Tyr and Trp) that we expected to be crucial for guiding the regioselectivity of the reaction. We performed a detailed quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) study on wild-type and mutant structures. The work shows that Trp is involved in key protein loop-loop interactions that affect the shape and size of the substrate binding pocket and its mutation has major long-range effects. By contrast, the Tyr residue is shown to guide the regio- and stereoselectivity by holding the substrate and ferryl oxidant in a specific orientation through hydrogen bonding and π-stacking interactions. Compelling evidence is found that the Tyr residue is involved in expelling the product from the binding pocket after the reaction is complete. It is shown that mutations where the hydrogen bonding network that involves the Tyr and Trp residues is disrupted lead to major changes in folding of the protein and the size and shape of the substrate binding pocket. Specifically, the Trp residue positions the amino acid side chains of Arg and Glu in specific orientations with substrate. As such, the P4H enzyme is a carefully designed protein with a subtle and rigid secondary structure that enables the binding of substrate, guides the regioselectivity, and expels product efficiently.

Enzymatic halogenation and haloperoxidation are unusual processes in biology; however, a range of halogenases and haloperoxidases exist that are able to transfer an aliphatic or aromatic C–H bond into C–Cl/C–Br. Haloperoxidases utilize hydrogen peroxide, and in a reaction with halides (Cl−/Br−), they react to form hypohalides (OCl−/OBr−) that subsequently react with substrate by halide transfer. There are three types of haloperoxidases, namely the iron-heme, nonheme vanadium, and flavin-dependent haloperoxidases that are reviewed here. In addition, there are the nonheme iron halogenases that show structural and functional similarity to the nonheme iron hydroxylases and form an iron(IV)-oxo active species from a reaction of molecular oxygen with α-ketoglutarate on an iron(II) center. They subsequently transfer a halide (Cl−/Br−) to an aliphatic C–H bond. We review the mechanism and function of nonheme iron halogenases and hydroxylases and show recent computational modelling studies of our group on the hectochlorin biosynthesis enzyme and prolyl-4-hydroxylase as examples of nonheme iron halogenases and hydroxylases. These studies have established the catalytic mechanism of these enzymes and show the importance of substrate and oxidant positioning on the stereo-, chemo- and regioselectivity of the reaction that takes place.

In this work we present the first computational study on the hectochlorin biosynthesis enzyme HctB, which is a unique three-domain halogenase that activates non-amino acid moieties tethered to an acyl-carrier, and as such may have biotechnological relevance beyond other halogenases. We use a combination of small cluster models and full enzyme structures calculated with quantum mechanics/molecular mechanics methods. Our work reveals that the reaction is initiated with a rate-determining hydrogen atom abstraction from substrate by an iron (IV)-oxo species, which creates an iron (III)-hydroxo intermediate. In a subsequent step the reaction can bifurcate to either halogenation or hydroxylation of substrate, but substrate binding and positioning drives the reaction to optimal substrate halogenation. Furthermore, several key residues in the protein have been identified for their involvement in charge-dipole interactions and induced electric field effects. In particular, two charged second coordination sphere amino acid residues (Glu223 and Arg245) appear to influence the charge density on the Cl ligand and push the mechanism toward halogenation. Our studies, therefore, conclude that nonheme iron halogenases have a chemical structure that induces an electric field on the active site that affects the halide and iron charge distributions and enable efficient halogenation. As such, HctB is intricately designed for a substrate halogenation and operates distinctly different from other nonheme iron halogenases.

Nitrogenases catalyse nitrogen fixation to ammonia on a multinuclear Fe‐Mo centre, but their mechanism and particularly the order of proton and electron transfer processes that happen during the catalytic cycle is still unresolved. Recently, a unique biomimetic mononuclear iron model was developed using tris(phosphine)borate (TPB) ligands that was shown to convert N2 into NH3. Herein, we present a computational study on the [(TPB)FeN2]− complex and describe its conversion into ammonia through the addition of electrons and protons. In particular, we tested the consecutive proton transfer on only the distal nitrogen atom or alternated protonation of the distal/proximal nitrogen. It is found that the lowest energy pathway is consecutive addition of three protons to the same site, which forms ammonia and an iron‐nitrido complex. In addition, the proton transfer step of complexes with the metal in various oxidation and spin states were tested and show that the pK a values of biomimetic mononuclear nitrogenase intermediates vary little with iron oxidation states. As such, the model gives several possible NH3 formation pathways depending on the order of electron/proton transfer, and all should be physically accessible in the natural system. These results may have implications for enzymatic nitrogenases and give insight into the catalytic properties of mononuclear iron centres.

After major surgery there are complex and diverse changes in the IGFs and IGFBPs. The effect of these changes on IGF bioavailability may significantly affect the therapeutic potential of IGF-I in this setting.