Assignment of protonated R-homocitrate in extracted FeMo-cofactor of nitrogenase via vibrational circular dichroism spectroscopy

Protonation of FeMo-cofactor (FeMo-co) is important for the process of substrate hydrogenation. Its structure has been clarified as Δ-Mo*Fe(7)S(9)C(R-homocit*)(cys)(Hhis) after the efforts of nearly 30 years, but it remains controversial whether FeMo-co is protonated or deprotonated with chelated ≡C − O(H) homocitrate. We have used protonated molybdenum(V) lactate 1 and its enantiomer as model compounds for R-homocitrate in FeMo-co of nitrogenase. Vibrational circular dichroism (VCD) spectrum of 1 at 1051 cm(−1) is attributed to ≡C − O(H) vibration, and molybdenum(VI) R-lactate at 1086 cm(−1) is assigned as ≡C − O(α-alkoxy) vibration. These vibrations set up labels for the protonation state of coordinated α-hydroxycarboxylates. The characteristic VCD band of NMF-extracted FeMo-co is assigned to ν(C − O(H)), which is based on the comparison of molybdenum(VI) R-homocitrate. Density functional theory calculations are consistent with these assignments. To the best of our knowledge, this is the first time that protonated R-homocitrate in FeMo-co is confirmed by VCD spectra.