Density Functional Theory Study into the Reaction Mechanism of Isonitrile Biosynthesis by the Nonheme Iron Enzyme ScoE

Abstract

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.

Probing the Mechanism of Isonitrile Formation by a Non-Heme Iron(II)-Dependent Oxidase/Decarboxylase

“… Following the HAT at C5, the relative energy of the iron-hydroxyl intermediate was calculated to be −42.3 kcal/mol. Upon closer analysis, we found no radical character on the substrate in line with previous observations . While small model calculations show the radical-based mechanism to also be favorable at −9.5 kcal/mol relative to CABA (Figure S23), a carbocation mechanism may also explain the formation of the iron–hydroxyl intermediate with more favorable energetics (Figure S14).…”

“…Although previous computational studies have been performed on ScoE, our calculations incorporate the recently elucidated crystallographic positioning of CABA (Figure S15). ,, We also expanded upon previous ScoE QM models by including backbone atoms of key residues, the second coordination sphere residue Thr145, and the backbone of Tyr135, which forms a hydrogen bond with CABA (Figure ). For the QM calculations, all sidechain atoms were allowed to move freely, while the backbone atoms were restrained to maintain their crystal structure positions.…”

Deciphering the Reaction Pathway of Mononuclear Iron Enzyme-Catalyzed N≡C Triple Bond Formation in Isocyanide Lipopeptide and Polyketide Biosynthesis

“…38 However, the decarboxylation is found to be a preferred pathway in de Visser's studies. 39 During the decarboxylation, our calculation suggests that the resulting Fe(III)OH species may act as an electron sink to facilitate CO 2 departure. Notably, the energy barrier of the OH-rebound is slightly higher than the calculated values of several Fe/2OG hydroxylases.…”

“…As suggested by the QM(UB3LYP/B1)/MM-optimized geometries of key species involved in the reaction (Figure 5C), the vinyl CH of 3 is well-positioned to the presumptive Fe(IV)O species, suggesting that 3 can undergo a HAT to form a radical intermediate (3′) through the σ-pathway which has been suggested in Fe/2OG enzymes 60−63 and agrees with de Visser's study. 39 On the other hand, that HAT undergoes the π-pathway is suggested by Liu's QM/MM study. 38 Our calculations show that this HAT step has an energy barrier of 9.9 kcal/mol which is similar to the previous studies on ScoE.…”

Using Computational Chemistry To Reveal Nature’s Blueprints for Single-Site Catalysis of C–H Activation

“…For ScoE, a recently discovered isonitrile-forming enzyme, many mechanisms were proposed prior to crystallographic characterization of the protein–substrate conformation. These mechanisms ranged from hydroxylation to desaturation and nitrogen activation. − However, recent computational and experimental studies based on the crystal structure have shed light on the mechanism, revealing key hydrogen bonding interactions between the substrate and the protein environment that orient the substrate , (see Figure ). This precise substrate orientation facilitates C5–H abstraction rather than at the weaker proximal N–H bond.…”