EPR and Electron Nuclear Double Resonance spectroscopies here characterize CO binding to the active-site A cluster of wild-type (WT) Acetyl-CoA Synthase (ACS) and two variants, F229W and F229A. The A-cluster binds CO to a proximal Ni (Nip) that bridges a [4Fe-4S] cluster and a distal Nid. An alcove seen in the ACS crystal structure near the A-cluster, defined by hydrophobic residues including F229, forms a cage surrounding a Xe mimic of CO. Previously, we only knew WT ACS bound a single CO to form the Ared-CO intermediate, containing Nip(I)-CO with CO located on the axis of the d z 2 odd-electron orbital (g⊥ > g|| ∼ 2). Here, the two-dimensional field-frequency pattern of 2K-35 GHz 13C-ENDOR spectra collected across the Ared-CO EPR envelope reveals a second CO bound in the d z 2 orbital’s equatorial plane. This WT A-cluster conformer dominates the nearly conservative F229W variant, but 13C-ENDOR reveals a minority “A” conformation with (g|| > g⊥ ∼ 2) characteristic of a “cloverleaf” (e.g., d x 2- y 2) odd-electron orbital, with Nip binding two, apparently “in-plane” CO. Disruption of the alcove through introduction of the smaller alanine residue in the F229A variant diminishes conversion to Ni­(I) ∼ 10-fold and introduces extensive cluster flexibility. 13C-ENDOR shows the F229A cluster is mostly (60%) in the “A” conformation but with ∼20% each of the WT conformer and an “O” state in which d z 2 Nip(I) (g⊥ > g|| ∼ 2) surprisingly lacks CO. This paper thus demonstrates the importance of an intact alcove in forming and stabilizing the Ni­(I)-CO intermediate in the Wood-Ljungdahl pathway of anaerobic CO and CO2 fixation.

The chapter focuses on the methods involved in producing and characterizing two key nickel–iron–sulfur enzymes in the Wood–Ljungdahl pathway (WLP) of anaerobic conversion of carbon dioxide fixation into acetyl-CoA: carbon monoxide dehydrogenase (CODH) and acetyl-CoA synthase (ACS). The WLP is used for biosynthesis of cell material and energy conservation by anaerobic bacteria and archaea, and it is central to several industrial biotechnology processes aimed at using syngas and waste gases for the production of fuels and chemicals. The pathway can run in reverse to allow organisms, e. g., methanogens and sulfate reducers, to grow on acetate. The CODH and ACS intertwine to form a tenacious CODH/ACS complex that converts CO2, a methyl group, and coenzyme A into acetyl-CoA. CODH also behaves as a modular unit that can function as an independent homodimer. Besides coupling to ACS, CODH can interact with hydrogenases to couple CO oxidation to H2 formation. These enzymes have been purified and characterized from several microbes.

[FeFe] hydrogenases are highly active catalysts for hydrogen conversion. Their active site has two components: a [4Fe−4S] electron relay covalently attached to the H 2 binding site and a diiron cluster ligated by CO, CN – , and 2-azapropane-1,3-dithiolate (ADT) ligands. Reduction of the [4Fe−4S] site was proposed to be coupled with protonation of one of its cysteine ligands. Here, we used time-resolved infrared (TRIR) spectroscopy on the [FeFe] hydrogenase from Chlamydomonas reinhardtii ( Cr HydA1) containing a propane-1,3-dithiolate (PDT) ligand instead of the native ADT ligand. The PDT modification does not affect the electron transfer step to [4Fe−4S] H but prevents the enzyme from proceeding further through the catalytic cycle. We show that the rate of the first electron transfer step is independent of the pH, supporting a simple electron transfer rather than a proton-coupled event. These results have important implications for our understanding of the catalytic mechanism of [FeFe] hydrogenases and highlight the utility of TRIR.

EPR and Electron Nuclear Double Resonance spectroscopies here characterize CO binding to the active-site A cluster of wild-type (WT) Acetyl-CoA Synthase (ACS) and two variants, F229W and F229A. The A-cluster binds CO to a proximal Ni (Nip) that bridges a [4Fe-4S] cluster and distal Nid. An alcove seen in the ACS crystal-structure near the A-cluster, defined by hydrophobic residues including F229, forms a cage surrounding a Xe mimic of CO and is suggested to 'cradle' this CO. Previously, we only knew WT ACS bound a single CO in the Ared-CO intermediate, here seen as forming Nip(I)-CO with CO on-axis of the dz 2 odd-electron orbital (g^>g||~2). The two-dimensional field-frequency pattern of 2K-35 GHz 13 C-ENDOR spectra collected across the Ared-CO EPR envelope now reveals a second CO bound in the dz 2 orbital's equatorial plane. This WT A-cluster conformer dominates the nearly-conservative F229W variant, but 13 C-ENDOR reveals a minority "A" conformation with (g||>g^~2

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 (Ni p ) within the A cluster of ACS undergoes major geometric and redox changes as it transits the planar Ni p , tetrahedral Ni p −CO and planar Ni p −Me and Ni p −Ac intermediates. We propose that the Ni p 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.