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 (Ni-p) that bridges a [4Fe-4S] cluster and a distal Ni-d. 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 A(red)-CO intermediate, containing Ni-p(I)-CO with CO located on the axis of the d(z)(2) odd-electron orbital (g(perpendicular to) > g(parallel to) similar to 2). Here, the two-dimensional field-frequency pattern of 2K-35 GHz C-13-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 C-13-ENDOR reveals a minority "A" conformation with (g(parallel to) > g(vertical bar) similar to 2) characteristic of a "cloverleaf" (e.g., d(x)(2) - (2)(y)) 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) similar to 10-fold and introduces extensive cluster flexibility. C-13-ENDOR shows the F229A cluster is mostly (60%) in the "A" conformation but with similar to 20% each of the WT conformer and an "O" state in which d(z)(2) Ni-p(I) (g(perpendicular to) > g(parallel to) similar to 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.
