¹³C ENDOR Characterization of the Central Carbon within the Nitrogenase Catalytic Cofactor Indicates That the CFe₆Core 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 (E 0; α-Val70) to those of (ii) α-Ile70, (iii) α-Ala70-substituted proteins; (iv) crystallographically characterized CO-inhibited “hi-CO” state; (v) E 4(4H) Janus intermediate, activated for N2 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 13C 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.

Understanding the Electronic Structure Basis for N2 Binding to FeMoco: A Systematic Quantum Mechanics/Molecular Mechanics Investigation

“…Clearly, this suggests that the coordination change of the binding site Fe is likely to accompany N 2 binding and we note that a flexible Fe–C interaction has indeed been suggested as a role for the interstitial carbide, with model compounds from Peters being particularly relevant in this context . However, recent 13 C ENDOR data of various states of FeMoco are more indicative of a stabilizing rather than flexible role for the interstitial carbide . In the diamagnetically substituted model, only the Fe and N 2 atoms were allowed to move and with no protons present either, and elongation of the Fe–C bond constitutes the only structural rearrangement possible.…”

“… 99 However, recent 13 C ENDOR data of various states of FeMoco are more indicative of a stabilizing rather than flexible role for the interstitial carbide. 100 In the diamagnetically substituted model, only the Fe and N 2 atoms were allowed to move and with no protons present either, and elongation of the Fe–C bond constitutes the only structural rearrangement possible.…”

The E3 state of FeMoco: one hydride, two hydrides or dihydrogen?

“…In fact, according to recent 13 C ENDOR studies of multiple trapped FeMoco states with labelled interstitial carbide, 112 the carbide hyperfine coupling is practically unchanged. A likely interpretation is that the carbide appears more likely to have a general stabilizing role 112 during catalysis. Together with the observation that most hybrid functionals also predict worse FeMoco structures (overestimating Fe-Fe, Fe-S and Fe-C distances) our interpretation is that only non-hybrid functionals and functionals with 0-10% HF exchange describe FeMoco qualitatively correctly.…”

Structural correlations of nitrogenase active sites using nuclear resonance vibrational spectroscopy and QM/MM calculations

“…Recently, Hoffman and co-workers have extensively employed ENDOR spectroscopy across multiple redox states and intermediates of Mo N 2 ase to establish that the carbide atom serves to maintain the structural rigidity of the cofactor (acting as a “heart of steel”). 31 Seven different protein samples were investigated, including both resting and reactive intermediate states, and the 13 C isotopic hyperfine coupling constants were found to range from −1.3 to +2.7 MHz, indicating negligible changes to the carbide structure for Mo N 2 ase during turnover. Meanwhile, FeVco was long believed to be a direct analog of FeMoco, with V replacing Mo at the apical position of the cluster, however, crystal structures of the VFe protein have unexpectedly and consistently revealed that one of three belt sulfurs of FeVco is replaced by a carbonate ligand.…”