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13C ENDOR Characterization of the Central Carbon within the Nitrogenase Catalytic Cofactor Indicates That the CFe6Core Is a Stabilizing “Heart of Steel”
- Source :
- Journal of the American Chemical Society; October 2022, Vol. 144 Issue: 40 p18315-18328, 14p
- Publication Year :
- 2022
-
Abstract
- Substrates and inhibitors of Mo-dependent nitrogenase bind and react at Fe ions of the active-site FeMo-cofactor [7Fe–9S–C–Mo–homocitrate] contained within the MoFe protein α-subunit. The cofactor contains a CFe6core, a carbon centered within a trigonal prism of six Fe, whose role in catalysis is unknown. Targeted 13C labeling of the carbon enables electron-nuclear double resonance (ENDOR) spectroscopy to sensitively monitor the electronic properties of the Fe–C bonds and the spin-coupling scheme adopted by the FeMo-cofactor metal ions. This report compares 13CFe6ENDOR measurements for (i) the wild-type protein resting state (E0; α-Val70) to those of (ii) α-Ile70, (iii) α-Ala70-substituted proteins; (iv) crystallographically characterized CO-inhibited “hi-CO” state; (v) E4(4H) Janus intermediate, activated for N2binding/reduction by accumulation of 4[e–/H+]; (vi) E4(2H)* state containing a doubly reduced FeMo-cofactor without Fe-bound substrates; and (vii) propargyl alcohol reduction intermediate having allyl alcohol bound as a ferracycle to FeMo-cofactor Fe6. All states examined, both S= 1/2 and 3/2 exhibited near-zero 13C isotropic hyperfine coupling constants, Ca= [−1.3 ↔ +2.7] MHz. Density functional theory computations and natural bond orbital analysis of the Fe−C bonds show that this occurs because a (3 spin-up/3 spin-down) spin-exchange configuration of CFe6Fe-ion spins produces cancellation of large spin-transfers to carbon in each Fe–C bond. Previous X-ray diffraction and DFT both indicate that trigonal-prismatic geometry around carbon is maintained with high precision in all these states. The persistent structure and Fe–C bonding of the CFe6core indicate that it does not provide a functionally dynamic (hemilabile) “beating heart”─instead it acts as “a heart of steel”, stabilizing the structure of the FeMo-cofactor-active site during nitrogenase catalysis.
Details
- Language :
- English
- ISSN :
- 00027863 and 15205126
- Volume :
- 144
- Issue :
- 40
- Database :
- Supplemental Index
- Journal :
- Journal of the American Chemical Society
- Publication Type :
- Periodical
- Accession number :
- ejs60906386
- Full Text :
- https://doi.org/10.1021/jacs.2c06149