Diverging Reaction Pathways and Key Intermediates in Ethylene Forming Enzyme.

Area of Science:

• Biochemistry
• Enzymology
• Computational Chemistry

Background:

• Ethylene-forming enzyme (EFE) is a unique non-heme iron(II)- and 2-oxoglutarate-dependent oxygenase.
• Unlike other oxygenases, EFE primarily produces CO2 and ethylene from 2-oxoglutarate, not succinate.
• Understanding EFE's catalytic mechanism is crucial for enzyme engineering and applications.

Purpose of the Study:

• To investigate the reaction pathways of ethylene formation (EF) in EFE.
• To elucidate the role of intermediates and branch points in the catalytic cycle.
• To revise the reaction mechanism of EFE based on computational and spectroscopic evidence.

Main Methods:

• Multifaceted computational approach: molecular dynamics, quantum mechanics/molecular mechanics (QM/MM).
• Theoretical Mössbauer spectroscopy to identify iron-containing species.
• Analysis of the protein's intrinsic electric field effects on reaction pathways.

Main Results:

• Identified a novel, earlier second branch point for ethylene formation (EF) and 3-hydroxypropionate pathways.
• Revealed multiple low-energy EF pathways involving Fe(II)-carbonates or Fe(II)-pyrocarbonates.
• Identified intermediates consistent with experimental Mössbauer spectroscopy data for Fe(II) species.

Conclusions:

• Proposed a revised reaction mechanism for ethylene formation in EFE, retaining C2-derived CO2.
• Highlighted the significance of the identified intermediates and branch points for EF pathway control.
• Findings provide a basis for modifying EFE to alter product yield in ethylene formation.