Invited Talk 21st International Conference on Biological Inorganic Chemistry 2025

Mechanistic Studies of the Ethylene-Forming Enzyme (122315)

Evan J Burke 1 , Rachelle A Copeland 1 , Jeffrey W Slater 1 , Chao Wang 1 , Shengbin Zhou 1 , Holly Lussier 1 , Katherine M Davis 1 , Elizabeth J Blaesi 1 , Tokufu K Shoda 1 , Yash Dixit 1 , Irene Schaperdoth 1 , Carsten Krebs 1 , J. Martin Bollinger, Jr. 1 , Amie K Boal 1 , Elvira R Sayfutyarova 1
  1. Pennsylvania State University, State College, PA, United States

The ethylene-forming enzyme (EFE) is a bifunctional Fe(II)- and 2-(oxo)glutarate (Fe/2OG)-dependent enzyme. In its minor reaction (~30%), it activates O2 at its Fe(II) center to couple the decarboxylation of 2OG to CO2 and succinate to the hydroxylation of C5 of the substrate, L-arginine. In its major reaction (~70%), which requires L-arginine only as effector, EFE activates O2 to promote the fragmentation of 2OG to three equivalents for CO2/(bi)carbonate derived from C1, C2, and C5 and ethylene derived from the C3-C4 dimethylene unit [1].

We have studied this reaction by a combination of biochemical, analytical (LC/MS and GC/MS), rapid-kinetic and spectroscopic (stopped-flow absorption, stopped-flow FTIR, and freeze-quench Mössbauer), X-ray crystallographic, and computational methods [2-5]. Our studies provide detailed insight into the mechanisms of EFE. They also revealed a third reaction, viz the generation of 3-hydroxy-propionate (3HP) derived from the C3-C5 fragment of 2OG. Our studies are consistent with a mechanism, wherein the Fe(II)-persuccinate complex is the branchpoint of the arginine-oxidizing and ethylene-forming reactions. The ethylene-forming path proceeds via a succinylperoxycarbonato intermediate, which can undergo O-O homolysis, followed by β-scission of the ensuing succinyl radical to generate a propionate-3-yl radical. Our results suggest that the latter intermediate is the second branchpoint, which can either undergo C4-C5 scission to yield ethylene or it can couple to the C1-derived (bi)carbonato ligand to generate a carbonate-monoester, which eliminates the alternative 3HP product. 

 

Financial support by the Department of Energy (DOE BES-DE-SC0016255) is gratefully acknowledged.

 

References

Fukuda, H.; Ogawa, T.; Tazaki, M.; Nagahama, K.; Fujiil, T.; Tanase, S.; Morino, Y. Biophys. Res. Commun. 1992, 188, 483–489.

Copeland, R. A.; Davis, K. M.; Shoda, T. K. C.; Blaesi, E. J.; Boal, A. K.; Krebs, C.; Bollinger, J. M., Jr. Am. Chem. Soc. 2021, 143, 2293–2303.

Copeland, R. A.; Zhou, S.; Schaperdoth, I.; Shoda, T. K. C.; Bollinger, J. M., Jr.; Krebs, C. Science 2021, 373, 1489–1493.

Burke, E. J.; Copeland, R. A.; Dixit, Y.; Krebs, C.; Bollinger, J. M., Jr. Am. Chem. Soc. 2024, 146, 1977–1983.

Burke, E. J.; Wang, C.; Slater, J. W.; Lussier, H.; Krebs, C., Bollinger, J. M., Jr., Sayfutyarova, E. R. in preparation