| Autor: |
James CD; Department of Chemistry, Northwestern University, Evanston, Illinois 60208, United States., Wiley S; Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606 United States., Ragsdale SW; Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606 United States., Hoffman BM; Department of Chemistry, Northwestern University, Evanston, Illinois 60208, United States. |
| Jazyk: |
angličtina |
| Zdroj: |
Journal of the American Chemical Society [J Am Chem Soc] 2020 Sep 09; Vol. 142 (36), pp. 15362-15370. Date of Electronic Publication: 2020 Aug 26. |
| DOI: |
10.1021/jacs.0c05950 |
| Abstrakt: |
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 ⊥ > g || ∼ 2). Here, the two-dimensional field-frequency pattern of 2K-35 GHz 13 C-ENDOR spectra collected across the A red -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 13 C-ENDOR reveals a minority "A" conformation with (g || > g ⊥ ∼ 2) characteristic of a "cloverleaf" (e.g., d x 2 - y 2 ) odd-electron orbital, with Ni p 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. 13 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 z 2 Ni p (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 2 fixation. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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