Mechanistic Insights into Acetyl CoA Synthase from Nickel-Substituted Azurin

CO dehydrogenase (CODH) and Acetyl CoA synthase (ACS) belong to the ancient Wood-Ljungdhal pathway, widely speculated to have been the first metabolic pathway to develop in the earliest lifeforms. Early life forms were strictly anaerobic and this metabolic pathway allowed those organisms to grow solely on CO and CO₂. CODH first catalyzes the reversible reduction of CO₂ to CO. ACS then catalyzes C-C bond formation between CO and CH₃ to form an acetyl intermediate which is subsequently added to Coenzyme A, forming the biological building-block, acetyl-CoA. As well as biological interest, the study of this efficient CO₂ fixation pathway has much relevance today for the role that it may play in fuel sustainability. The CODH/ACS pathway provides a mechanism for the reduction of CO₂ into liquid fuels, aiding the design of synthetic catalysts with broad environmental applications. 

Elucidating the mechanism of CODH/ACS has proved difficult due to the low protein expression, poor metal incorporation, and extreme oxygen sensitivity of the enzymes. Their study is further complicated by the presence of multiple metal sites, producing spectroscopic signals that can be difficult to deconvolute. In particular, the A cluster of ACS contains a total of 6 metal atoms: a distal Ni, a proximal Ni, and a 4Fe-4S cluster. Thus, the idea of generating active site mimics in small protein models rose to the forefront. These efforts to mimic the A cluster of ACS have focused on the proximal Ni, predicted to be the site of CO and CH₃ binding. Significant progress on the mechanism of ACS has been made by studying a Ni-substituted azurin model which was able to coordinate both ACS substrates, generate an acetyl intermediate, and complete thioester bond formation with CoA. A thorough characterization of the Ni electronic states at intermediate points in the catalysis revealed a methyl bound intermediate with an inverted ligand field postulated as a reactivity gating mechanism for native ACS. The results of this mechanistic probe also favor the proposal of a paramagnetic mechanism through the Ni^I/Ni^III couple for native ACS and solves the debate over ordered vs. disordered substrate binding. The results of this study not only further our understanding of these ancient enzymes but have provided a roadmap for the design and engineering of other metalloenzymes and model proteins. 

Related literature:

Can et. al. Chem. Rev. 2014, 114, 4149−4174

Manesis et. al. J. Am. Chem. Soc. 2017, 139, 10328−10338

Kisgeropoulos et. al. J. Am. Chem. Soc. 2021, 143, 849−867

Manesis, Yerbulekova et. al. Proc. Natl. Acad. Sci. USA 2022, 119, e2123022119