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".
J Am Chem Soc
; 142(36): 15362-15370, 2020 09 09.
Article
em En
| MEDLINE
| ID: mdl-32786751
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 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 dz2 odd-electron orbital (g⥠> g|| â¼ 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 dz2 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⥠⼠2) characteristic of a "cloverleaf" (e.g., dx2-y2) 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) â¼ 10-fold and introduces extensive cluster flexibility. 13C-ENDOR shows the F229A cluster is mostly (60%) in the "A" conformation but with â¼20% each of the WT conformer and an "O" state in which dz2 Nip(I) (g⥠> g|| â¼ 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.
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Base de dados:
MEDLINE
Assunto principal:
Acetilcoenzima A
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Monóxido de Carbono
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Ressonância Magnética Nuclear Biomolecular
Tipo de estudo:
Prognostic_studies
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article