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We thank Alvaro Salinero-Lanzarote for performing cell growth and grants from the U.S. Department of Energy (DOE) , Office of Science, Office of Basic Energy Sciences supported genetic studies, protein production, trapping of states for study, and ENDOR spectroscopy (DE-SC0010687, DE-SC0010834, and DE-SC0019342 to L.C.S., D.R.D., and B.M.H.) . Support for S.R. was provided by the U.S. DOE, Office of Science, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences. Computer time was provided by the National Energy Research Scientific Computing Center (NERSC) , a U.S. DOE Office of Science User Facility operated by Lawrence Berkeley National Laboratory, and the Molecular Sciences Computing Facility (MSCF) in the Environmental Molecular Sciences Laboratory, a DOE User Facility located at the Pacific Northwest National Laboratory (PNNL) . PNNL is operated by Battelle for the DOE under Contract number DE-AC05-76RL01830.

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C-13 ENDOR Characterization of the Central Carbon within the Nitrogenase Catalytic Cofactor Indicates That the CFe6 Core Is a Stabilizing "Heart of Steel"

Publicado en:Journal Of The American Chemical Society. 144 (40): 18315-18328 - 2022-09-27 144(40), DOI: 10.1021/jacs.2c06149

Autores: Lukoyanov, Dmitriy A.; Yang, Zhi-Yong; Perez-Gonzalez, Ana; Raugei, Simone; Dean, Dennis R.; Seefeldt, Lance C.; Hoffman, Brian M.;

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Resumen

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 alpha-subunit. The cofactor contains a CFe6 core, a carbon centered within a trigonal prism of six Fe, whose role in catalysis is unknown. Targeted C-13 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 (CFe6)-C-13 ENDOR measurements for (i) the wild-type protein resting state (E-0; alpha-Val(70)) to those of (ii) alpha-Ile(70), (iii) alpha-Ala(70)-substituted proteins; (iv) crystallographically characterized CO-inhibited "hi-CO" state; (v) E-4(4H) Janus intermediate, activated for N-2 binding/reduction by accumulation of 4[e(-)/H+]; (vi) E-4(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 C-13 isotropic hyperfine coupling constants, (C)a = [-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 CFe6 Fe-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 CFe6 core 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.

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