C 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 (Ni) that bridges a [4Fe-4S] cluster and a distal Ni. 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-CO intermediate, containing Ni(I)-CO with CO located on the axis of the d odd-electron orbital (g > g ∼ 2). Here, the two-dimensional field-frequency pattern of 2K-35 GHz C-ENDOR spectra collected across the A-CO EPR envelope reveals a second CO bound in the d orbital's equatorial plane. This WT A-cluster conformer dominates the nearly conservative F229W variant, but C-ENDOR reveals a minority "A" conformation with (g > g ∼ 2) characteristic of a "cloverleaf" (e.g., d-) odd-electron orbital, with Ni 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. C-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 Ni(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 CO fixation.