Ligand Field Inversion as a Mechanism to Gate Bioorganometallic Reactivity: Investigating a Biochemical Model of Acetyl CoA Synthase Using Spectroscopy and Computation

Abstract

The biological global carbon cycle is largely regulated through microbial nickel enzymes, including carbon monoxide dehydrogenase (CODH), acetyl coenzyme A synthase (ACS), and methyl coenzyme M reductase (MCR). These systems are suggested to utilize organometallic intermediates during catalysis, though characterization of these species has remained challenging. We have established a mutant of nickel-substituted azurin as a scaffold upon which to develop protein-based models of enzymatic intermediates, including the organometallic states of ACS. In this work, we report the comprehensive investigation of the S = 1/2 Ni–CO and Ni–CH₃ states using pulsed EPR spectroscopy and computational techniques. While the Ni–CO state shows conventional metal–ligand interactions and a classical ligand field, the Ni–CH₃ hyperfine interactions between the methyl protons and the nickel indicate a closer distance than would be expected for an anionic methyl ligand. Structural analysis instead suggests a near-planar methyl ligand that can be best described as cationic. Consistent with this conclusion, the frontier molecular orbitals of the Ni–CH₃ species indicate a ligand-centered LUMO, with a d⁹ population on the metal center, rather than the d⁷ population expected for a typical metal–alkyl species generated by oxidative addition. Collectively, these data support the presence of an inverted ligand field configuration for the Ni–CH₃ Az species, in which the lowest unoccupied orbital is centered on the ligands rather than the more electropositive metal. These analyses provide the first evidence for an inverted ligand field within a biological system. The functional relevance of the electronic structures of both the Ni–CO and Ni–CH₃ species are discussed in the context of native ACS, and an inverted ligand field is proposed as a mechanism by which to gate reactivity both within ACS and in other thiolate-containing metalloenzymes.