Poster Presentation 21st International Conference on Biological Inorganic Chemistry 2025

Understanding how the outer sphere modulates carbon-carbon bond formation in an artificial metalloenzyme mimic of acetyl coenzyme A synthase (#486)

Kathryn G. Woodburn 1 , Kevin Enrique Rivera Cruz 1 , Isaiah E. Ervin 1 , Hannah S. Shafaat 1
  1. Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, CA, United States

Despite sustained efforts to reduce anthropogenic greenhouse gas emissions, the climate crisis remains, necessitating continued research into innovative carbon recycling solutions.1 For inspiration, we turn to the Wood-Ljungdahl pathway, the oldest known carbon dioxide (CO2) fixation pathway in which cornerstone enzymes carbon monoxide dehydrogenase (CODH) and acetyl coenzyme A synthase (ACS) produce the metabolic building block acetyl-coenzyme A (CoA) from cationic methyl species (CH3), CO2, and CoA.2,3 Once CODH reduces CO2 to carbon monoxide (CO), CO travels through an intramolecular tunnel to ACS for incorporation into acetyl-CoA.4 Recent efforts to characterize ACS catalysis assert an “alcove” of hydrophobic secondary (2°) sphere residues moderates substrate behavior.5 Because oxygen sensitivity stymies ACS practical applications, an alternative nickel-substituted azurin (NiAz) mutant has been developed as a geometric and electronic mimic of ACS that can produce a thioester from C1 starting materials.6,7 While lacking an alcove of hydrophobic residues, our proposed NiAz substrate channel is gated by a phenylalanine (F) 15 residue. Analogous to ACS, we hypothesized the substrate channel modulates the activity of our artificial metalloenzyme. To test this, we designed a series of 2° sphere NiAz mutants probing how F15 mutations impact substrate binding. Alkyl binding kinetics reveal wide variation in substrate binding rates, suggesting F15 critically impacts channel structure and establishing premise for 2° sphere stabilization of transient intermediates. Electron paramagnetic resonance spectroscopy CO binding studies with calculations demonstrate F15X mutants retain comparable electronics to ACS but differ in binding energies. Overall, differences in mutant reactivity illustrate how 2° sphere mutations can be leveraged to improve the capacity of NiAz as a biocatalyst for C1-upconversion chemistry. Ultimately, understanding how Nature designs proteins to control gaseous substrates via intramolecular tunnels can accelerate rational design of artificial metalloenzymes for C1-upgrading chemistry.

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