Recasting Nitrogenase's Carbide Role as a Beating Heart of Steel: A Joint Inorganic and Organic Perspective for...

Nitrogenase's cofactor features a trigonal prismatic interstitial carbide, an architectural motif without parallel in other biological systems whose enzymatic significance remains unclear. A ¹³C ENDOR study indicated negligible hyperfine coupling at the carbide, hinting at an inert geometric and electronic structure that preserves its trigonal prismatic framework and the antiferromagnetic coupling of the Fe-sites. Contrary to the "heart of steel" interpretation, the "beating heart" model proposes that structural flexibility aids cofactor stabilization during catalysis. Here, we establish the theoretical foundation of the carbide's bonding using valence bond (VB) and molecular orbital (MO) theory, both indicating a preference for the trigonal prismatic geometry with antiferromagnetic coupling. The carbide in FeMoco's resting state shows six equivalent σ-bonds with half bond order from sp²-hybridization, linking insights from inorganic and organic chemistry. Our findings, supported by broken-symmetry density functional theory (BS-DFT) and quantum mechanics/molecular mechanics (QM/MM) modeling, show that the carbide retains its trigonal prismatic geometry, while its local σ/π bonding and hybridization adapt to the spin coupling of the Fe-centers. Altogether, our findings suggest that the carbide imparts an inert geometric framework alongside a dynamic electronic structure that enables the catalytic reduction of diverse substrates.