Thioester synthesis by a designed nickel enzyme models prebiotic energy conversion

Abstract

The formation of carbon–carbon bonds from prebiotic precursors such as carbon dioxide represents the foundation of all primordial life processes. In extant organisms, this reaction is carried out by the carbon monoxide dehydrogenase (CODH)/acetyl coenzyme A synthase (ACS) enzyme, which performs the cornerstone reaction in the ancient Wood–Ljungdahl metabolic pathway to synthesize the key biological metabolite, acetyl-CoA. Despite its significance, a fundamental understanding of this transformation is lacking, hampering efforts to harness analogous chemistry. To address these knowledge gaps, we have designed an artificial metalloenzyme within the azurin protein scaffold as a structural, functional, and mechanistic model of ACS. We demonstrate the intermediacy of the Ni I species and requirement for ordered substrate binding in the bioorganometallic carbon–carbon bond-forming reaction from the one-carbon ACS substrates. The electronic and geometric structures of the nickel-acetyl intermediate have been characterized using time-resolved optical, electron paramagnetic resonance, and X-ray absorption spectroscopy in conjunction with quantum chemical calculations. Moreover, we demonstrate that the nickel-acetyl species is chemically competent for selective acyl transfer upon thiol addition to biosynthesize an activated thioester. Drawing an analogy to the native enzyme, a mechanism for thioester generation by this ACS model has been proposed. The fundamental insight into the enzymatic process provided by this rudimentary ACS model has implications for the evolution of primitive ACS-like proteins. Ultimately, these findings offer strategies for development of highly active catalysts for sustainable generation of liquid fuels from one-carbon substrates, with potential for broad applications across diverse fields ranging from energy storage to environmental remediation.

How to Build a Metalloenzyme: Lessons from a Protein-Based Model of Acetyl Coenzyme A Synthase

“…Addition of thiolate to the Ni II −C(O)CH 3 Az species resulted in the detection of large quantities of S-methylthioacetate, demonstrating the formation of a C−S bond through thiolysis of the nickel-acetyl species (Figure 5B). The high yields of thioester formed coupled with the low amounts of acetate observed, which could derive from either direct hydrolysis of the unstable thioester or slow hydrolysis of the Ni−C(O)CH 3 species, 4,48 favor selective C−S bond formation in the nickel azurin model, further reproducing the reactivity of native ACS.…”

“…Guided by the principles of rational design and electronic structure analysis, we have been able to reconstruct ACS reactivity within a small metalloprotein model. 4 This firstgeneration system sets biochemical precedence for many of the postulated but never-observed species in ACS, allowing us to resolve possible mechanistic details that are backed by established biochemical feasibility. For example, even with a large excess of low-potential reductant (E′ ≤ −1 V vs NHE), the Ni 0 state is inaccessible within the Az scaffold, as evidenced by a lack of any change in the Ni I Az optical or EPR intensities.…”

“…Given the key electronic similarities between M121A NiAz and ACS, we can assess the possibility of downstream reactivity by introducing both ACS substrates. The addition of CO to Ni II –CH 3 Az reveals the formation of a new species with Ni II -based spectral signatures (Figure A), which were characterized using optical and X-ray spectroscopy . Nickel K-edge extended absorption fine structure (EXAFS) analysis of this species indicates that, in addition to retaining coordination by a cysteine thiolate ligand and two histidine nitrogens, the structure can be accurately modeled by the addition of a third, light-atom scatterer to give a four-coordinate structure.…”

“…The addition of CO to Ni II − CH 3 Az reveals the formation of a new species with Ni II -based spectral signatures (Figure 5A), which were characterized using optical and X-ray spectroscopy. 4 Nickel K-edge extended absorption fine structure (EXAFS) analysis of this species indicates that, in addition to retaining coordination by a cysteine thiolate ligand and two histidine nitrogens, the structure can be accurately modeled by the addition of a third, light-atom scatterer to give a four-coordinate structure. Analysis of the X-ray absorption near edge spectroscopy (XANES) data in conjunction with DFT calculations leads to assignment of this new species as a high-spin, trigonal pyramidal Ni II −C(O)CH 3 Az state.…”

“…19,54 Finally, the observation of near-stoichiometric rather than catalytic amounts of thioester synthesis in the NiAz model suggests an irreversible degradation process following thiolysis, likely associated with demetalation of the resultant Ni 0 species. 4 Whether this irreversible inactivation can be circumvented through the incorporation of a redox shuttle remains to be seen. If so, investigating structure−function relationships of the modified NiAz model will reveal the required properties of this redox shuttle, including reduction potential, intramolecular electron transfer rates, and electronic coupling, which may lead to identification of an analogous shuttle in native ACS.…”