Cryo-annealing of Photoreduced CdS Quantum Dot–Nitrogenase MoFe Protein Complexes Reveals the Kinetic Stability of the E4(2N2H) Intermediate

A critical step in the mechanism of N2 reduction to 2NH3 catalyzed by the enzyme nitrogenase is the reaction of the four-electron/four-proton reduced intermediate state of the active-site FeMo-cofactor (E4(4H)). This state is a junction in the catalytic mechanism, either relaxing by the reaction of a metal bound Fe-hydride with a proton forming H2 or going forward with N2 binding coupled to the reductive elimination (re) of two Fe-hydrides as H2 to form the E4(2N2H) state. E4(2N2H) can relax to E4(4H) by the oxidative addition (oa) of H2 and release of N2 or can be further reduced in a series of catalytic steps to release 2NH3. If the H2re/oa mechanism is correct, it requires that oa of H2 be associative with E4(2N2H). In this report, we have taken advantage of CdS quantum dots in complex with MoFe protein to achieve photodriven electron delivery in the frozen state, with cryo-annealing in the dark, to reveal details of the E-state species and to test the stability of E4(2N2H). Illumination of frozen CdS:MoFe protein complexes led to formation of a population of reduced intermediates. Electron paramagnetic resonance spectroscopy identified E-state signals including E2 and E4(2N2H), as well as signals suggesting the formation of E6 or E8. It is shown that in the frozen state when pN2 is much greater than pH2, the E4(2N2H) state is kinetically stable, with very limited forward or reverse reaction rates. These results establish that the oa of H2 to the E4(2N2H) state follows an associative reaction mechanism.

ABSTRACT: A critical step in the mechanism of N 2 reduction to 2NH 3 catalyzed by the enzyme nitrogenase is the reaction of the four-electron/four-proton reduced intermediate state of the active-site FeMo-cofactor (E 4 (4H)).This state is a junction in the catalytic mechanism, either relaxing by the reaction of a metal bound Fe-hydride with a proton forming H 2 or going forward with N 2 binding coupled to the reductive elimination (re) of two Fe-hydrides as H 2 to form the E 4 (2N2H) state.E 4 (2N2H) can relax to E 4 (4H) by the oxidative addition (oa) of H 2 and release of N 2 or can be further reduced in a series of catalytic steps to release 2NH 3 .If the H 2 re/oa mechanism is correct, it requires that oa of H 2 be associative with E 4 (2N2H).In this report, we have taken advantage of CdS quantum dots in complex with MoFe protein to achieve photodriven electron delivery in the frozen state, with cryo-annealing in the dark, to reveal details of the E-state species and to test the stability of E 4 (2N2H).Illumination of frozen CdS:MoFe protein complexes led to formation of a population of reduced intermediates.Electron paramagnetic resonance spectroscopy identified Estate signals including E 2 and E 4 (2N2H), as well as signals suggesting the formation of E 6 or E 8 .It is shown that in the frozen state when pN 2 is much greater than pH 2 , the E 4 (2N2H) state is kinetically stable, with very limited forward or reverse reaction rates.These results establish that the oa of H 2 to the E 4 (2N2H) state follows an associative reaction mechanism.
N itrogenases are two-component enzyme systems that catalyze the ATP-dependent reduction of N 2 to ammonia (NH 3 ) and hydrogen (H 2 ). 1 Mo-nitrogenase is the most well-studied of the three nitrogenase systems (i.e., Mo-, V-, and Fe-nitrogenase), from which an understanding of the stepwise proton-and electron-transfer steps has evolved into a model of the N 2 reduction scheme. 1−3 The Fe protein component of MoFe-nitrogenase catalyzes the ATP-dependent delivery of electrons to the MoFe protein component, 4,5 which harbors the iron−molybdenum cofactor (FeMo-co), which is the site of N 2 binding and activation (Figure 1).Under ideal conditions, the catalytic cycle requires a minimum of eight electron-transfer steps from Fe protein to MoFe protein, producing two molecules of NH 3 and one molecule of H 2 from one N 2 , eight protons, and 16 ATP. 5,6−10 A key aspect of the model for the N 2 reduction reaction is that the resting state FeMo-co, E 0 , must be reduced by four electrons and four proteins to form E 4 (4H), the precursor to N 2 binding and activation. 9−12 A release of H 2 from E 4 (4H) by reductive elimination (re) of two hydrides energetically drives the binding of N 2 and formation of the E 4 (2N2H) intermediate (Figure 1). 11,12Therefore, the reversible conversion of E 4 (4H) to E 4 (2N2H) is a critical point in the catalytic mechanism between the forward pathway to NH 3 formation and the backward pathway of H 2 release.The reversible reaction involves the oxidative addition (oa) of H 2 to E 4 (2N2H) that re-forms E 4 (4H).The dependence of re/oa kinetics on the pN 2 /pH 2 ratio led to the prediction that E 4 (2N2H) is kinetically stable as N 2 increases or if H 2 decreases.Trapping reactions under different N 2 partial pressures have been used to demonstrate that the decay of E 4 (2N2H) is slower as pN 2 increases 6 or when pH 2 is lowered by flushing reactions under argon. 10,11Collectively, the results support that the pN 2 /pH 2 ratio and the re/oa equilibrium control E 4 (2N2H) stability (Figure 1).However, H 2 coproduction by nitrogenase under turnover prevents complete elimination of H 2 and a determination of whether oa of H 2 follows an associative versus dissociative process.
−20 The physical coupling of nanocrystal materials and MoFe protein enables electron delivery under illumination at different temperatures. 17,20llumination of reactions in the frozen state (∼233 K) has been used to photoaccumulate and trap P cluster or FeMo-co cluster intermediates in the absence of catalytic turnover. 18,19herefore, this approach might be useful for generating and testing the stability of catalytic intermediates in the absence of turnover and H 2 production.Herein, CdS quantum dot (QD)-MoFe protein biohybrids were prepared and illuminated in reactions at 233 K under 1 atm of N 2 , followed by cryoannealing in the dark at 236 K (see Supporting Information for details).Changes in populations of even E-states (Figure 1) in MoFe protein under annealing can be monitored by electron paramagnetic resonance (EPR) spectroscopy for observing the relaxation kinetics of intermediates and analyzing the stability of the E 4 (2N2H) state.
Figure 2 displays the EPR spectra time course used to monitor the E-state signal intensities.−24 In addition, the simulation revealed signals consistent with those previously assigned to E 6 /E 8 (Table S2).Annealing of the sample at 236 K in the dark, which prevents additional photoexcited electron transfer, led to some changes in signal intensities in the S = 3/2 and S = 1/2 regions of the EPR spectra (Figure 2).Simulations of each spectral time point were used to determine the E-state populations (Figure S1 and S2, Tables S3 and S4), which are plotted in Figure 3. Dark annealing led to a cumulative decrease in E 2 (2H)1b, E 2 (2H)1c, and E 4 (4H) attributed to the backward reaction, H 2 release (Figure 1), which coincided with an increase in E 0 .Thus, the annealing of CdS:MoFe protein complexes at 236 K did not inhibit the hydride protonation at FeMo-co.In addition to these changes, signals matching those for the E 6 /E 8 states also attenuated (Table S4).The S = 3/2 region spectra (colored traces, T = 3.6 K, P = 1 mW) with callouts for g-values of E 0 , E 2 (2H)1b, and E 2 (2H)1c signals obtained from simulations (gray).Bottom: The S = 1/2 region spectra (colored traces, T = 12 K, P = 1 mW) with simulations (gray) (see Figure S2 and Table S2).The inset shows intensity changes of the g x components of the E 4 (2N2H) (g x = 2.095) and E 4 (H) (g x = 2.17 and g x = 2.14) signals.See the Supporting Information for experimental details, simulation methods, and simulated signals (Figures S1 and S2 and Table S1).
To assess the decay kinetics of the E-states, the time course plots in Figure 3 were fit to the stretched exponential equation (eq 1): where E n (t) is the E-state population at time t, A is the value of E n (t) at t = 0, τ is the time constant for change in E n (t), and m is the breadth of the distribution, i.e., 0 < m ≤ 1 (fit values are in Table S5). 25,26The decline in the cumulative populations of E 2 , E 4 (4H), and E 6 /E 8 (τ = 90 min) was ∼10-fold higher than accumulation of E 0 (τ = 1263 min) (Figure 3, Tables S3 and S4), a difference that may result from other internal processes, such as P cluster oxidation (τ = 0.22) by electron transfer to FeMo-co that reduces the population of E 0 .Most strikingly, there was no net loss in the E 4 (2N2H) population throughout the annealing time course (Figure 3).Under the conditions created here, there should be minimal formation of H 2 from hydride protonation.Given that the rate of decay of the E 4 (2N2H) state back to the E 4 (4H) state is a second-order reaction, the combination of pN 2 = 1 atm with a low pH 2 (i.e., pN 2 ≫pH 2 ) is expected to result in a very low rate of oa (Figure 1).Consistent with this expectation is the observed slow decay of E 4 (2N2H), with a measured τ = 3.6 × 10 6 min (Table S5). 10,11The net effect of the conditions created by the delivery of electrons to the MoFe protein in the frozen state using CdS nanoparticles is the stabilization of the E 4 (2N2H) state.These findings reveal that the oa reaction between E 4 (2N2H) and H 2 is an associative rather than a dissociative process in which H 2 binds to the E 4 (2N2H) state, rather than N 2 being lost before H 2 binds.
The understanding of the N 2 reduction reaction by nitrogenases has largely developed from studies on the complete biological two-component system, where the requirement of ATP-dependent electron delivery by Fe protein places constraints on testing kinetic models in the absence of turnover.We have demonstrated that nanocrystals and lightcontrolled electron delivery can bypass some of these constraints to gain new insights into N 2 reduction by nitrogenase.Here, CdS:MoFe protein biohybrids were used to clearly demonstrate the kinetic stability of the mechanistically central N 2 bound state, E 4 (2N2H).The results give new insights into the nitrogenase mechanism, providing direct experimental evidence to support the assertion from theory that the E 4 (2N2H) state is stable at low concentrations of H 2 , 12 revealing that the oa requires H 2 binding to the E 4 (2N2H) state rather than release of N 2 followed by H 2 binding.

Figure 1 .
Figure 1.(A).Natural and biohybrid systems used for delivery of electrons to the nitrogenase MoFe protein.Electron delivery by Fe protein (blue) to MoFe protein (gray and purple) is coupled to repeated cycles of ATP binding and hydrolysis at 298 K. Photoexcited CdS quantum dots (orange) couple photon absorption to electron delivery, which can be performed across a range of ambient to subambient (i.e., 233 K) temperatures (D, donor; D ox , oxidized donor).(B).Modified Lowe−Thorneley scheme of the N 2 reduction reaction; it is not the full scheme which includes N 2 binding to E 3 . 7,13−15 Stepwise proton and electron delivery to the MoFe protein FeMo-co site leads to reduction of E 0 to form E n -states.E 4 (4H) binds N 2 by reductive elimination (re) of H 2 to form E 4 (2N2H).Kinetic stability of the E 4 (2N2H) intermediate is under control of the reaction product, H 2 , and E 4 (2N2H) can convert back to E 4 (4H) by oxidative addition (oa) of H 2 and release of N 2 .(C).Photoexcited electron delivery by CdS in the frozen state under 1 atm of N 2 (left) limits MoFe protein turnover (TON) and H 2 production compared with ambient reaction temperatures with Fe protein (right).
Materials and methods for CdS synthesis, MoFe protein isolation, EPR spectroscopy spectral simulations and spin quantifications; figures showing the S = 3/2 (T = 3.6 K, P = 1 mW) and S = 1/2 (T = 12 K, P = 1 mW) EPR spectra collected for CdS:MoFe protein for each dark annealing time step and spectral simulations; tables of EPR signal g-values and the values from fits to stretched exponential eq 1 (PDF)