Mechanism of N₂ Reduction Catalyzed by Fe-Nitrogenase Involves Reductive Elimination of H₂

Of the three forms of nitrogenase (Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase), Fe-nitrogenase has the poorest ratio of N₂ reduction relative to H₂ 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 H₂ is a key feature in the N₂ reduction mechanism. The N₂ 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 N₂ 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 N₂ reduction. The fully functioning Fe-nitrogenase system was found to have specific activities for N₂ reduction (1 atm) of 181 ± 5 nmol NH₃ min^–1 mg^–1 FeFe protein, for proton reduction (in the absence of N₂) of 1085 ± 41 nmol H₂ min^–1 mg^–1 FeFe protein, and for acetylene reduction (0.3 atm) of 306 ± 3 nmol C₂H₄ min^–1 mg^–1 FeFe protein. Under turnover conditions, N₂ reduction is inhibited by H₂ and the enzyme catalyzes the formation of HD when presented with N₂ and D₂. 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 N₂ reduction occurring by reductive elimination of H₂

Evidence That the P_i Release Event Is the Rate-Limiting Step in the Nitrogenase Catalytic Cycle

Nitrogenase reduction of dinitrogen (N₂) to ammonia (NH₃) involves a sequence of events that occur upon the transient association of the reduced Fe protein containing two ATP molecules with the MoFe protein that includes electron transfer, ATP hydrolysis, P_i release, and dissociation of the oxidized, ADP-containing Fe protein from the reduced MoFe protein. Numerous kinetic studies using the nonphysiological electron donor dithionite have suggested that the rate-limiting step in this reaction cycle is the dissociation of the Fe protein from the MoFe protein. Here, we have established the rate constants for each of the key steps in the catalytic cycle using the physiological reductant flavodoxin protein in its hydroquinone state. The findings indicate that with this reductant, the rate-limiting step in the reaction cycle is not protein–protein dissociation or reduction of the oxidized Fe protein, but rather events associated with the P_i release step. Further, it is demonstrated that (i) Fe protein transfers only one electron to MoFe protein in each Fe protein cycle coupled with hydrolysis of two ATP molecules, (ii) the oxidized Fe protein is not reduced when bound to MoFe protein, and (iii) the Fe protein interacts with flavodoxin using the same binding interface that is used with the MoFe protein. These findings allow a revision of the rate-limiting step in the nitrogenase Fe protein cycle

The Electron Bifurcating FixABCX Protein Complex from <i>Azotobacter vinelandii</i>: Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis

The biological reduction of dinitrogen (N₂) to ammonia (NH₃) by nitrogenase is an energetically demanding reaction that requires low-potential electrons and ATP; however, pathways used to deliver the electrons from central metabolism to the reductants of nitrogenase, ferredoxin or flavodoxin, remain unknown for many diazotrophic microbes. The FixABCX protein complex has been proposed to reduce flavodoxin or ferredoxin using NADH as the electron donor in a process known as electron bifurcation. Herein, the FixABCX complex from <i>Azotobacter vinelandii</i> was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (<i>E</i>_m = −320 mV) coupled to reduction of flavodoxin semiquinone (<i>E</i>_m = −460 mV) and reduction of coenzyme Q (<i>E</i>_m = 10 mV). Knocking out <i>fix</i> genes rendered Δ<i>rnf A. vinelandii</i> cells unable to fix dinitrogen, confirming that the FixABCX system provides another route for delivery of electrons to nitrogenase. Characterization of the purified FixABCX complex revealed the presence of flavin and iron–sulfur cofactors confirmed by native mass spectrometry, electron paramagnetic resonance spectroscopy, and transient absorption spectroscopy. Transient absorption spectroscopy further established the presence of a short-lived flavin semiquinone radical, suggesting that a thermodynamically unstable flavin semiquinone may participate as an intermediate in the transfer of an electron to flavodoxin. A structural model of FixABCX, generated using chemical cross-linking in conjunction with homology modeling, revealed plausible electron transfer pathways to both high- and low-potential acceptors. Overall, this study informs a mechanism for electron bifurcation, offering insight into a unique method for delivery of low-potential electrons required for energy-intensive biochemical conversions