Identification of a Key Catalytic Intermediate Demonstrates That Nitrogenase Is Activated by the Reversible Exchange of N₂ for H₂

Abstract

Freeze-quenching nitrogenase during turnover with N2 traps an S = ½ intermediate that was shown by ENDOR and EPR spectroscopy to contain N2 or a reduction product bound to the active-site molybdenum-iron cofactor (FeMo-co). To identify this intermediate (termed here EG), we turned to a quench-cryoannealing relaxation protocol. The trapped state is allowed to relax to the resting E0 state in frozen medium at a temperature below the melting temperature; relaxation is monitored by periodically cooling the sample to cryogenic temperature for EPR analysis. During −50°C cryoannealing of EG prepared under turnover conditions in which the concentrations of N2 and H2 ([H2], [N2]) are systematically and independently varied, the rate of decay of EG is accelerated by increasing [H2] and slowed by increasing [N2] in the frozen reaction mixture; correspondingly, the accumulation of EG is greater with low [H2] and/or high [N2]. The influence of these diatomics identifies EG as the key catalytic intermediate formed by reductive elimination (re) of H2 with concomitant N2 binding, a state in which FeMo-co binds the components of diazene (an N-N moiety, perhaps N2 and two [e−/H+] or diazene itself). This identification combines with an earlier study to demonstrate that nitrogenase is activated for N2 binding and reduction through the thermodynamically and kinetically reversible reductive-elimination/oxidative-addition exchange of N2 and H2, with an implied limiting stoichiometry of eight electrons/protons for the reduction of N2 to two NH3.

Reductive Elimination of H2 Activates Nitrogenase to Reduce the N≡N Triple Bond: Characterization of the E4(4H) Janus Intermediate in Wild-Type Enzyme

“…In this case its concentration relative to that of E 4 (2N2H) is governed by the equilibrium of Fig 3 , when, as expected, the forward and reverse steps of the re/oa equilibrium are rapid compared to the slow delivery of the next electron from Fe red ( Fig 1 ), $$\frac{E_4\left(2N2H\right)}{E_4\left(4H\right)}=K_{re}\frac{P\left(N_2\right)}{P\left(H_2\right)}\propto P\left(N_2\right)$$ Here K re is the equilibrium constant for the re/oa equilibrium, and a simple proportionality to P(N 2 ) follows from our observation that at these high enzyme concentrations, turnover produces a roughly constant (saturating) concentration of H 2 regardless of P(N 2 ). 15 Not only does this P(N 2 ) dependence supports the idea that the g 1 = 2.15 state is indeed the WT E 4 (4H), but of central importance to this study, the ability to prepare samples whose dominant EPR signal is that of the g 1 = 2.15 state enables its characterization and identification, as now described.…”

“…In this case we can rewrite eq 2 to approximate K re as, $$K_{re}=\frac{E_4\left(2N2H\right)}{E_4\left(4H\right)}\cdot \frac{P\left(H_2\right)}{P\left(N_2\right)}$$ The N 2 partial pressure is fixed by the experimental conditions; earlier observations suggest that saturating concentrations of H 2 are formed during turnover under all P(N 2 ), which suggests an effective P(H 2 ) ~ 1atm. 15 As a result, one obtains: $$K_{re}~true(~\frac{3}{2}true)\ast true(\frac{~1}{0.05}true)~30$$ $${\Delta }_{re}{G}^0=-RT\ln \left(K_{ro}\right)~-2kcal∕mol$$ The LT kinetic measurements likewise yielded values for the re process: K re ~ 0.7 and Δ re G 0 ~ +0.2 kcal/mol. 2,4 Given the difference in methodologies – direct observation of species in equilibrium in the present study, analysis of turnover kinetics in the former – and the differences in origin of the MoFe proteins – Azotobacter vinelandii in the present study and Klebsiella pneumoniae in the former - we consider the measurements to be in excellent agreement: nitrogenase catalysis, driven by the re of H 2 , turns the highly endothermic first step in the reduction of the N 2 triple bond, (to the diazene level) into the essentially thermoneutral re/oa equilibrium conversion of Fig 3 .…”

“…EPR conditions: for E 4 (4H) spectra the same as in Fig 4 ; for E 4 (2N2H), E 2 and E 0 the same as used in previous studies. 15 …”

“…14 More recently, we identified an S = ½ EPR signal that appears during N 2 reduction by wild type (WT) MoFe protein as arising from FeMo-co of the E 4 (2N2H) state formed by re of H 2 with N 2 reduction, Fig 3 , and in so doing confirmed the prediction that the ( re/oa ) activation equilibrium is not only thermodynamically, but also kinetically reversible. 15 Characterization of the E 4 (4H) ‘Janus’ intermediate as carrying four reducing equivalents in the form of two [Fe-H-Fe] bridging hydrides thus laid the foundation for the re/oa mechanism, Fig 3 , with its obligatory formation of one H 2 per N 2 reduced and resultant limiting stoichiometry of eq 1 , 15 while the success of predictions based on the mechanism provides powerful support for the mechanism.…”

Reversible Photoinduced Reductive Elimination of H2 from the Nitrogenase Dihydride State, the E4(4H) Janus Intermediate

“…This in turn implies the limiting stoichiometry of eight electrons/protons for the reduction of N 2 to two NH 3 (eq 1). 16 …”