An alcove at the acetyl-CoA synthase nickel active site is required for productive substrate CO binding and anaerobic carbon fixation.

One of the seven natural CO2 fixation pathways, the anaerobic Wood-Ljungdahl pathway (WLP) is unique in generating CO as a metabolic intermediate, operating through organometallic intermediates, and in conserving (versus utilizing) net ATP. The key enzyme in the WLP is acetyl-CoA synthase (ACS), which uses an active site [2Ni-4Fe-4S] cluster (A-cluster), a CO tunnel, and an organometallic (Ni-CO, Ni-methyl, and Ni-acetyl) reaction sequence to generate acetyl-CoA. Here, we reveal that an alcove, which interfaces the tunnel and the A-cluster, is essential for CO2 fixation and autotrophic growth by the WLP. In vitro spectroscopy, kinetics, binding, and in vivo growth experiments reveal that a Phe229A substitution at one wall of the alcove decreases CO affinity thirty-fold and abolishes autotrophic growth; however, a F229W substitution enhances CO binding 80-fold. Our results indicate that the structure of the alcove is exquisitely tuned to concentrate CO near the A-cluster; protect ACS from CO loss during catalysis, provide a haven for inhibitory CO, and stabilize the tetrahedral coordination at the Nip site where CO binds. The directing, concentrating, and protective effects of the alcove explain the inability of F209A to grow autotrophically. The alcove also could help explain current controversies over whether ACS binds CO and methyl through a random or ordered mechanism. Our work redefines what we historically refer to as the metallocenter "active site". The alcove is so crucial for enzymatic function that we propose it is part of the active site. The community should now look for such alcoves in all "gas handling" metalloenzymes.

Characterization of Methyl- and Acetyl-Ni Intermediates in Acetyl CoA Synthase Formed during Anaerobic CO2 and CO Fixation.

The Wood-Ljungdahl Pathway is a unique biological mechanism of carbon dioxide and carbon monoxide fixation proposed to operate through nickel-based organometallic intermediates. The most unusual steps in this metabolic cycle involve a complex of two distinct nickel-iron-sulfur proteins: CO dehydrogenase and acetyl-CoA synthase (CODH/ACS). Here, we describe the nickel-methyl and nickel-acetyl intermediates in ACS completing the characterization of all its proposed organometallic intermediates. A single nickel site (Nip) within the A cluster of ACS undergoes major geometric and redox changes as it transits the planar Nip, tetrahedral Nip-CO and planar Nip-Me and Nip-Ac intermediates. We propose that the Nip intermediates equilibrate among different redox states, driven by an electrochemical-chemical (EC) coupling process, and that geometric changes in the A-cluster linked to large protein conformational changes control entry of CO and the methyl group.

Crystal structure of bis(3,5-dichloro-2-hydroxybenzyl)(2-methoxyethyl)amine.

The title compound, systematic name 4,4',6,6'-tetrachloro-2,2'-{[(2-methoxy-ethyl)azanediyl]bis(methylene)}diphenol (C17H17Cl4NO, 1), was prepared via a modified Mannich reaction between 2-meth-oxy-ethyl-amine, 2,4-di-chloro-phenol, and aqueous formaldehyde. The resulting amine bis-(phenol) provides an inter-esting comparison to related species as a result of the electron-withdrawing substituents on the phenol rings, in combination with similar steric parameters. One of the Cl atoms was modeled as a two-component disorder with partial occupancies of 0.49 (3) and 0.51 (3), while the pendant ether group was modeled as a two-component disorder with partial occupancies of 0.867 (3) and 0.133 (3). A comparison of metrical parameters for the title compound and closely related structures provides insight into the use of these species as ligands to support transition-metal complexes for applications as homogeneous catalysts.