Search

Nitrogenase catalyzes the reduction of dinitrogen (N2) to two ammonia (NH3) at its active site FeMocofactor 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 the 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 oxidoreductase enzymes and to biomimetic complexes. [ABSTRACT FROM AUTHOR]

Investigations of reduction of nitrite (NO2–) to ammonia (NH) by nitrogenase indicate a limiting stoichiometry, NO2– + 6e– + 12ATP + 7H+ → NH3 + 2H2O + 12ADP + 12Pi. Two intermediates freeze-trapped during NO2– turnover by nitrogenase variants and investigated by Q-band ENDOR/ESEEM are identical to states, denoted H and I, formed on the pathway of N2 reduction. The proposed NO2– reduction intermediate hydroxylamine (NH2OH) is a nitrogenase substrate for which the H and I reduction intermediates also can be trapped. Viewing N2 and NO2– reductions in light of their common reduction intermediates and of NO2– reduction by multiheme cytochrome c nitrite reductase (ccNIR) leads us to propose that NO2– reduction by nitrogenase begins with the generation of NO2H bound to a state in which the active-site FeMo-co (M) has accumulated two [e–/H+] (E2), stored as a (bridging) hydride and proton. Proton transfer to NO2H and H2O loss leaves M–[NO+]; transfer of the E2 hydride to the [NO+] directly to form HNO bound to FeMo-co is one of two alternative means for avoiding formation of a terminal M–[NO] thermodynamic “sink”. The N2 and NO2– reduction pathways converge upon reduction of NH2NH2 and NH2OH bound states to form state H with [−NH2] bound to M. Final reduction converts H to I, with NH bound to M. The results presented here, combined with the parallels with ccNIR, support a N2 fixation mechanism in which liberation of the first NH occurs upon delivery of five [e–/H+] to N2, but a total of seven [e–/H+] to FeMo-co when obligate H2 evolution is considered, and not earlier in the reduction process. [ABSTRACT FROM AUTHOR]

Of the three forms of nitrogenase (Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase), Fe-nitrogenase has the poorest ratio of N 2 reduction relative to H 2 evolution. Recent work on the Mo-nitrogenase has revealed that reductive elimination of two bridging Fe-H-Fe hydrides on the active site FeMo-cofactor to yield H 2 is a key feature in the N 2 reduction mechanism. The N 2 reduction mechanism for the Fe-nitrogenase active site FeFe-cofactor was unknown. Here, we have purified both component proteins of the Fe-nitrogenase system, the electron-delivery Fe protein (AnfH) plus the catalytic FeFe protein (AnfDGK), and established its mechanism of N 2 reduction. Inductively coupled plasma optical emission spectroscopy and mass spectrometry show that the FeFe protein component does not contain significant amounts of Mo or V, thus ruling out a requirement of these metals for N 2 reduction. The fully functioning Fe-nitrogenase system was found to have specific activities for N 2 reduction (1 atm) of 181 ± 5 nmol NH 3 min -1 mg -1 FeFe protein, for proton reduction (in the absence of N 2 ) of 1085 ± 41 nmol H 2 min -1 mg -1 FeFe protein, and for acetylene reduction (0.3 atm) of 306 ± 3 nmol C 2 H 4 min -1 mg -1 FeFe protein. Under turnover conditions, N 2 reduction is inhibited by H 2 and the enzyme catalyzes the formation of HD when presented with N 2 and D 2 . These observations are explained by the accumulation of four reducing equivalents as two metal-bound hydrides and two protons at the FeFe-cofactor, with activation for N 2 reduction occurring by reductive elimination of H 2 .

Nitrogenase catalyzes the reduction of dinitrogen (N 2 ) to two ammonia (NH 3 ) at its active site FeMo-cofactor through a mechanism involving reductive elimination of two [Fe-H-Fe] bridging hydrides to make H 2 . A competing reaction is the protonation of the hydride [Fe-H-Fe] to make H 2 . 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 the MoFe protein allowing examination of the mechanism of H 2 formation by the metal-hydride protonation reaction. The ratio of catalytic current in mixtures of H 2 O and D 2 O, the proton inventory, was found to change linearly with the D 2 O/H 2 O ratio, revealing that a single H/D is involved in the rate-limiting step of H 2 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 H 2 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 H 2 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 H 2 formation by hydride protonation, but also illustrates a strategy for mechanistic study that can be applied to other oxidoreductase enzymes and to biomimetic complexes.