13C Electron Nuclear Double Resonance Spectroscopy Shows Acetyl-CoA Synthase Binds Two Substrate CO in Multiple Binding Modes and Reveals the Importance of a CO-Binding 'Alcove'

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 dz2 odd-electron orbital (g{perp}>g||[~]2). The two-dimensional field-frequency pattern of 2K-35 GHz 13C-ENDOR spectra collected across the Ared-CO EPR envelope now reveals a second CO bound in the dz2 orbitals equatorial plane. This WT A-cluster conformer dominates the nearly-conservative F229W variant, but 13C-ENDOR reveals a minority "A" conformation with (g||>g{perp}[~]2) characteristic of a cloverleaf (eg. dx2-y2) odd-electron orbital, and 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) [~]tenfold 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 dz2 Nip(I) (g{perp}>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.