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The nonheme iron enzyme ScoE catalyzes the biosynthesis of an isonitrile substituent in a peptide chain. To understand details of the reaction mechanism we created a large active site cluster model of 212 atoms that contains substrate, the active oxidant and the first- and second-coordination sphere of the protein and solvent. Several possible reaction mechanisms were tested and it is shown that isonitrile can only be formed through two consecutive catalytic cycles that both use one molecule of dioxygen and α-ketoglutarate. In both cycles the active species is an iron(IV)-oxo species that in the first reaction cycle reacts through two consecutive hydrogen atom abstraction steps: first from the N–H group and thereafter from the C–H group to desaturate the NH-CH2 bond. The alternative ordering of hydrogen atom abstraction steps was also tested but found to be higher in energy. Moreover, the electronic configurations along that pathway implicate an initial hydride transfer followed by proton transfer. We highlight an active site Lys residue that is shown to donate charge in the transition states and influences the relative barrier heights and bifurcation pathways. A second catalytic cycle of the reaction of iron(IV)-oxo with desaturated substrate starts with hydrogen atom abstraction followed by decarboxylation to give isonitrile directly. The catalytic cycle is completed with a proton transfer to iron(II)-hydroxo to generate the iron(II)-water resting state. The work is compared with experimental observation and previous computational studies on this system and put in a larger perspective of nonheme iron chemistry.

A recently characterized cytochrome P450 isozyme GcoA activates lignin components through a selective O‐demethylation or alternatively an acetal formation reaction. These are important reactions in biotechnology and, because lignin is readily available; it being the main component in plant cell walls. In this work we present a density functional theory study on a large active site model of GcoA to investigate syringol activation by an iron(IV)‐oxo heme cation radical oxidant (Compound I) leading to hemiacetal and acetal products. Several substrate‐binding positions were tested and full energy landscapes calculated. The study shows that substrate positioning determines the product distributions. Thus, with the phenol group pointing away from the heme, an O‐demethylation is predicted, whereas an initial hydrogen‐atom abstraction of the weak phenolic O‐H group would trigger a pathway leading to ring‐closure to form acetal products. Predictions on how to engineer P450 GcoA to get more selective product distributions are given., Using an unnatural natural product: Density functional theory calculations on a large cluster model of cytochrome P450 GcoA show that the enzyme can bind and activate lignin monomer units and convert them into hemiacetal and acetal products. Suggestions on how to bioengineer the enzyme to improve the selectivity are given.

A density functional theory study is presented here to offer mechanistic insights and explications of experimentally intriguing observations in the Au(I)-catalyzed cyclization of cyclic and acyclic acetals of alkynylaldehydes that leads to indenone formation. The reactivity of catalytic cycles with and without methoxy migration is clearly defined when the alkyne terminus is phenylated. The reaction mechanism of indenone formation proceeds first with the coordination of Au(I) to alkyne to initiate the reaction with 1,5-H shift as a rate-determining step (RDS), and the fastest 1,5-H shift is achieved when one phenyl ring carries an electron-donating group and the other one is substituted with an electron-withdrawing group. Following the 1,5-H shift, the reaction undergoes feasible steps that are cyclization and 1,2-H shift before elimination to persist the iterative cycle, but the reactivity of both steps is highly affected by the existence of the phenyl group on the alkyne terminus. The unreactivity of the alkyne terminus not bearing a phenyl ring is because the cyclization is thermodynamically disfavorable, subsequently deactivating the 1,2-H shift kinetically and thermodynamically. The absence of a tether in the acetal unit considerably outpaces any 1,5-H shift and instead activates 1,5-methoxy migration, giving methoxy-migrated indenone, with the 1,2-OMe shift being an RDS.

Cytochrome P450 enzymes are versatile biocatalysts found in most forms of life. Generally, the cytochrome P450s react with dioxygen and hence are haem-based mono-oxygenases; however, in specific isozymes, H2O2 rather than O2 is used and these P450s act as peroxygenases. The P450 OleTJE is a peroxygenase that binds long to medium chain fatty acids and converts them to a range of products originating from Cα-hydroxylation, Cβ-hydroxylation, Cα-Cβ desaturation and decarboxylation of the substrate. There is still controversy regarding the details of the reaction mechanism of P450 OleTJE; how the products are formed and whether the product distributions can be influenced by external perturbations. To gain further insights into the structure and reactivity of P450 OleTJE, we set up a range of large active site model complexes as well as full enzymatic structures and did a combination of density functional theory studies and quantum mechanics/molecular mechanics calculations. In particular, the work focused on the mechanisms leading to these products under various reaction conditions. Thus, for a small cluster model, we find a highly selective Cα-hydroxylation pathway that is preferred over Cβ-H hydrogen atom abstraction by at least 10 kcal mol-1. Introduction of polar residues to the model, such as an active site protonated histidine residue or through external electric field effects, lowers the Cβ-H hydrogen atom abstraction barriers are lowered, while a full QM/MM model brings the Cα-H and Cβ-H hydrogen atom abstraction barriers within 1 kcal mol-1. Our studies; therefore, implicate that environmental effects in the second-coordination sphere can direct and guide selectivities in enzymatic reaction mechanisms.

The nonheme iron dioxygenase 2-(trimethylammonio)-ethylphosphonate dioxygenase (TmpA) is an enzyme involved in the regio- and chemoselective hydroxylation at the C 1 -position of the substrate as part of the biosynthesis of glycine betaine in bacteria and carnitine in humans. To understand how the enzyme avoids breaking the weak C 2 -H bond in favor of C 1 -hydroxylation, we set up a cluster model of 242 atoms representing the first and second-coordination sphere of the metal center and substrate binding pocket and investigated possible reaction mechanisms of substrate activation by an iron(IV)-oxo species by density functional theory methods. In agreement with experimental product distributions, the calculations predict a favorable C 1 -hydroxylation pathway. The calculations show that the selectivity is guided through electrostatic perturbations inside the protein from charged residues, external electric fields and electric dipole moments. In particular, charged residues influence and perturb the homolytic bond strength of the C 1 -H and C 2 -H bonds of the substrate, and strongly strengthens the C 2 -H bond in the substrate-bound orientation.

The nonheme iron enzyme OrfP reacts with L‐Arg selectively to form the 3 R ,4 R ‐dihydroxyarginine product, which in mammals can inhibit the nitric oxide synthase enzymes involved in blood pressure control. To understand the mechanisms of dioxygen activation of L‐Arg by OrfP and how it enables two sequential oxidation cycles on the same substrate, we performed a density functional theory study on a large active site cluster model. We show that substrate binding and positioning in the active site guides a highly selective reaction through C 3 ‐H hydrogen atom abstraction. This happens despite the fact that the C 3 ‒H and C 4 ‒H bond strengths of L‐Arg are very similar. Electronic differences in the two hydrogen atom abstraction pathways drive the reaction with an initial C 3 ‒H activation to a low‐energy 5 s‐pathway, while substrate positioning destabilizes the C 4 ‒H abstraction and sends it over the higher‐lying 5 p‐pathway. We show that substrate and monohydroxylated products are strongly bound in the substrate binding pocket and hence product release is difficult and consequently its lifetime will be long enough to trigger a second oxygenation cycle.

Compost tea is gaining much interest due to its nutritional, biostimulation and disease suppression benefits to the plants. Four experiments were carried out to determine the effect of incubation temperature, incubation periods, dilution rate and nutritional sources on the microbial populations, physico-chemical properties and indole acetic acid (IAA) levels. Incubation at 28°C showed the highest number of bacteria and aerobic N2-fixing bacteria (ANFB), along with the highest levels of total nitrogen and IAA. All the microbial populations increased in proportion with increase in the incubation period; however, increasing the incubation more than 48 h did not show any significant improvement (P ≤ 0.05) in the physico-chemical properties or IAA levels. All microbial numbers and chemical properties decreased by increasing the dilution rate. Adding a mixture of the three used nutrients (molasses "0.5% v/v", ammonium nitrate "0.5 g/L" and di-potassium phosphate "0.5 g/L") resulted in the highest microbial populations (except for ANFB) as compared to the molasses, or double the amount of each other chemical alone. This study show the optimum conditions for preparing compost tea with high microbiological and physico-chemical properties. The study includes ANFB, important for soil application, and IAA, important for foliar application, which have not been studied before in compost tea. Key words: Compost tea extraction, factors, microbiology, physico-chemical, indole acetic acid (IAA), aerobic N2-fixing bacteria (ANFB).