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

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 CFe₆ core, 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 ¹³CFe₆ ENDOR 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 N₂ binding/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 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.

SUBMITTER: Lukoyanov DA

PROVIDER: S-EPMC10712652 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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

Lukoyanov Dmitriy A DA Yang Zhi-Yong ZY Pérez-González Ana A Raugei Simone S Dean Dennis R DR Seefeldt Lance C LC Hoffman Brian M BM

Journal of the American Chemical Society 20220927 40

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 CFe6 core, 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 ...[more]

PMID: 36166637

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Project description:N(2) binds to the active-site metal cluster in the nitrogenase MoFe protein, the FeMo-cofactor ([7Fe-9S-Mo-homocitrate-X]; FeMo-co) only after the MoFe protein has accumulated three or four electrons/protons (E(3) or E(4) states), with the E(4) state being optimally activated. Here we study the FeMo-co (57)Fe atoms of E(4) trapped with the α-70(Val→Ile) MoFe protein variant through use of advanced ENDOR methods: 'random-hop' Davies pulsed 35 GHz ENDOR; difference triple resonance; the recently developed Pulse-Endor-SaTuration and REcovery (PESTRE) protocol for determining hyperfine-coupling signs; and Raw-DATA (RD)-PESTRE, a PESTRE variant that gives a continuous sign readout over a selected radiofrequency range. These methods have allowed experimental determination of the signed isotropic (57)Fe hyperfine couplings for five of the seven iron sites of the reductively activated E(4) FeMo-co, and given the magnitude of the coupling for a sixth. When supplemented by the use of sum-rules developed to describe electron-spin coupling in FeS proteins, these (57)Fe measurements yield both the magnitude and signs of the isotropic couplings for the complete set of seven Fe sites of FeMo-co in E(4). In light of the previous findings that FeMo-co of E(4) binds two hydrides in the form of (Fe-(μ-H(-))-Fe) fragments, and that molybdenum has not become reduced, an 'electron inventory' analysis assigns the formal redox level of FeMo-co metal ions in E(4) to that of the resting state (M(N)), with the four accumulated electrons residing on the two Fe-bound hydrides. Comparisons with earlier (57)Fe ENDOR studies and electron inventory analyses of the bio-organometallic intermediate formed during the reduction of alkynes and the CO-inhibited forms of nitrogenase (hi-CO and lo-CO) inspire the conjecture that throughout the eight-electron reduction of N(2) plus 2H(+) to two NH(3) plus H(2), the inorganic core of FeMo-co cycles through only a single redox couple connecting two formal redox levels: those associated with the resting state, M(N), and with the one-electron reduced state, M(R). We further note that this conjecture might apply to other complex FeS enzymes.

Dataset Information

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

Publications

<sup>13</sup>C ENDOR Characterization of the Central Carbon within the Nitrogenase Catalytic Cofactor Indicates That the CFe<sub>6</sub> Core Is a Stabilizing "Heart of Steel".

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