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’

Abstract

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

Two ligand-binding sites in CO-reducing V nitrogenase reveal a general mechanistic principle

“…S6 (8)] (43). Note that a subpopulation of this enzyme also binds a second CO to its nickel site (44). With two further reduction steps to a terminal hydride at Fe6 [fig.…”