Diverging Reaction Pathways and Key Intermediates in Ethylene Forming Enzyme

Ethylene-forming enzyme (EFE) is a non-heme iron (II)- and 2-oxoglutarate-(Fe(II)/2OG)-dependent oxygenase that exhibits distinct catalytic activity. While most Fe(II)/2OG-dependent oxygenases catalyze the substrate hydroxylation accompanied by the decarboxylation of the 2OG cosubstrate to succinate, EFE primarily converts the 2OG into CO2 and ethylene. Experimental studies suggest that the reaction mechanism of EFE does not involve the Fe(IV)=O (ferryl) intermediate central in the consensus mechanism for hydroxylation in Fe(II)/2OG-dependent oxygenases, and the ethylene-forming reaction pathway diverges early in the catalytic cycle after binding molecular oxygen. In this paper, we employ a multifaceted approach, including molecular dynamics, quantum mechanics and molecular mechanics methods, theoretical Mössbauer spectroscopy, and the analysis of the intrinsic electric field exerted by the protein environment, to examine possible reaction pathways for ethylene formation and hydroxylation in EFE. Our study reveals a new reaction pathway with a low energy barrier via the formation of Fe(II)-pyrocarbonates, which is different from all previously proposed reaction mechanisms. Based on our results, we introduce a revised reaction mechanism for the ethylene formation in the EFE that is consistent with the available experimental data. This work also provides new insights into both the first and second branchpoints of the ethylene-forming pathway that can be useful in EFE modifications aimed at shifting the product yield in the EF reaction.