Mechanism of Nitrogenase H₂ Formation by Metal-Hydride Protonation Probed by Mediated Electrocatalysis and H/D Isotope Effects

Abstract

Nitrogenase catalyzes the reduction of dinitrogen (N2) to two ammonia (NH3) at its active site FeMo-cofactor through a mechanism involving reductive elimination of two [Fe-H-Fe] bridging hydrides to make H2. A competing reaction is the protonation of the hydride [Fe-H-Fe] to make H2. The overall nitrogenase rate-limiting step is associated with ATP-driven electron delivery from Fe protein, precluding isotope effect measurements on substrate reduction steps. Here, we use mediated bioelectrocatalysis to drive electron delivery to MoFe protein allowing examination of the mechanism of H2 formation by the metal-hydride protonation reaction. The ratio of catalytic current in mixtures of H2O and D2O, the proton inventory, was found to change linearly with the D2O/H2O ratio, revealing that a single H/D is involved in the rate limiting step of H2 formation. Kinetic models, along with measurements that vary the electron/proton delivery rate and use different substrates, reveal that the rate-limiting step under these conditions is the H2 formation reaction. Altering the chemical environment around the active site FeMo-cofactor in the MoFe protein either by substituting nearby amino acids or transferring the isolated FeMo-cofactor into a different peptide matrix, changes the net isotope effect, but the proton inventory plot remains linear, consistent with an unchanging rate-limiting step. Density functional theory predicts a transition state for H2 formation where the S-H+ bond breaks and H+ attacks the Fe-hydride, and explains the observed H/D isotope effect. This study not only reveals the nitrogenase mechanism of H2 formation by hydride protonation, but also illustrates a strategy for mechanistic study that can be applied to other enzymes and to biomimetic complexes.

Large Hydrogen Isotope Fractionation Distinguishes Nitrogenase-Derived Methane from Other Methane Sources

“…7; (74)). It is also possible that proton tunneling, which is generally thought to have a very large kinetic isotope effect (but also see 73,74) and has been proposed to occur in nitrogenase (82), could be contributing to the observed KIE, though we note that the temperature effect observed here is opposite of the predicted effect for tunneling (83,84).…”

“…αH2O/H2 of Mo-nitrogenase(74). For example, it could be that the V-and Fe-only nitrogenases are less selective for1 H compared to the Mo-nitrogenase.…”

Carbon Dioxide Insertion into Bridging Iron Hydrides: Kinetic and Mechanistic Studies

“…There are few reports detailing the kinetic parameters for hydride transfer from a transition metal center to CO 2 . Independent of this dearth of detailed kinetic studies, two mechanistic pathways are proposed: normal insertion wherein an H – attacks the π* orbital of CO 2 , and abnormal insertion in which a metallocarboxylate is generated , . The latter pathway is typically invoked for CO 2 reduction to CO and H 2 O, whereas the former leads to formate.…”

Natural and Engineered Electron Transfer of Nitrogenase

“…Hu et al also adopted this approach to demonstrate that the MoFe and FeFe proteins could reduce CO 2 to HCOO − with respective FEs of 9% and 32%. Khadka et al also demonstrated that this approach could be employed to investigate the rate-limiting step of the MoFe when catalyzing H + reduction [170]. In this study, the cobaltocene concentration was titrated such that electron transfer to the MoFe protein was not limiting, and bioelectrocatalytic H + reduction to H 2 was evaluated as a function of catalytic current.…”