Dissecting Electronic-Structural Transitions in the Nitrogenase MoFe Protein P-Cluster during Reduction

Abstract

The [8Fe-7S] P-cluster of nitrogenase MoFe protein mediates electron transfer from nitrogenase Fe protein during the catalytic production of ammonia. The P-cluster transitions between three oxidation states, P^N, P⁺, P²⁺ of which P^N↔P⁺ is critical to electron exchange in the nitrogenase complex during turnover. To dissect the steps in formation of P⁺ during electron transfer, photochemical reduction of MoFe protein at 231–263 K was used to trap formation of P⁺ intermediates for analysis by EPR. In complexes with CdS nanocrystals, illumination of MoFe protein led to reduction of the P-cluster P²⁺ that was coincident with formation of three distinct EPR signals: S = 1/2 axial and rhombic signals, and a high-spin S = 7/2 signal. Under dark annealing the axial and high-spin signal intensities declined, which coincided with an increase in the rhombic signal intensity. A fit of the time-dependent changes of the axial and high-spin signals to a reaction model demonstrates they are intermediates in the formation of the P-cluster P⁺ resting state and defines how spin-state transitions are coupled to changes in P-cluster oxidation state in MoFe protein during electron transfer.

Nitrogenase is a two-component enzyme that catalyzes the conversion of dinitrogen to ammonia. Under ideal reaction conditions, the Mo-dependent form of nitrogenase, composed of Fe protein and MoFe protein, catalyzes N₂ reduction to ammonia according to eq 1:¹

1

During turnover, the electrons required for N₂ reduction are transferred from Fe protein to MoFe protein, which is an α₂β₂ tetramer that coordinates two sets of unique metal clusters. The [8Fe-7S] P-cluster functions in electron transfer with Fe protein, and the [7Fe-9S-1Mo-C-Homocitrate] iron–molybdenum cofactor (FeMo-co) functions as the site of N₂ reduction.²

One of the unique aspects of how nitrogenase catalyzes ammonia production is the electron transfer process.^3,4 In the catalytic cycle, the P-cluster forms a metastable intermediate oxidation state, P⁺ ([7Fe^IIFe^III-7S]⁺¹), that is rapidly reduced (k > 1700 s^–1)³ during electron transfer. In addition to P⁺, the P-cluster forms two stable oxidation states, P^N ([8Fe^II-7S]⁰ and P²⁺ ([6Fe^II2Fe^III-7S]⁺²) (Figure 1).⁵ Transitions between P-cluster states involve extensive structural changes, including a switch in Fe-coordination of the central sulfide (S₁) that bridges the two [4Fe-3S] subclusters, and amide nitrogen coordination to Fe₅ by α-88 cysteine (α-88Cys) and β-188 serine (β-188Ser) oxygen coordination to Fe₆. Recently, the X-ray structure of MoFe protein was solved with the P-cluster poised in the P⁺ state, with an intermediate structural arrangement between P²⁺ and P^N (Figure 1).⁶ In the P⁺ state, the S₁ sulfide is pentacoordinate and β-188Ser coordinates Fe₆. The observation of structural changes in the MoFe protein P-cluster has been incorporated into conformational gating⁷ and mechanical coupling^8,9 electron transfer models. The model predicts that motions near the P-cluster and β-188Ser are coupled to “switch regions” in the Fe protein that steer structural interactions within the nitrogenase complex to enable electron delivery.⁹

Figure 1.

MoFe protein P-cluster oxidation state structures for P²⁺, P⁺, and P^N. The α-88^Cys (P²⁺↔P⁺) and β-188^Ser (P⁺↔P^N) ligands that undergo redox-coupled coordination changes to the P-cluster are shown. The P²⁺↔P⁺ transition involves exchange of the S₁–Fe₅ thiolate bond (gray arrow) and α-88^Cys amide bond at Fe₅ (blue arrow) and proceeds via proton-coupled electron transfer (PCET), where changes in bonding (box) may lead to different conformers during electron transfer. The P⁺↔P^N transition involves exchange of the S₁–Fe₆ thiolate bond and β-188^Ser serine hydroxylate bond at Fe₆ (red arrow). E_m = −309 mV at pH 8 for both transitions.¹¹ PDB Codes: P²⁺, 2MIN; P⁺, 6CDK; P^N, 3MIN.

The structural rearrangements of the P-cluster in different oxidation states also coincide with changes in spin states and EPR properties. P²⁺ is an integer spin state, likely S = 4, and gives rise to an EPR signal at g = 11.8,^10,11 whereas P^N is an S = 0 spin state and EPR-silent. The P⁺ oxidation state has a rhombic, S = 1/2 EPR signal at g = 2.05, 1.94, 1.81 that shifts to g = 2.03, 1.97, 1.93 when β-188Ser is substituted by Cys.^12,13 Additional magnetic signals associated with the P⁺ oxidation state include an S = 1/2 signal with g = 2.00 and 1.89,^13,14 and low-field S = 5/2 signals^12,13 (Table S1). Variations in P⁺ magnetic states have been observed in MoFe protein under different redox titration conditions (Table S2).¹³⁻¹⁵ Whether these states have a functional role in electron transfer in MoFe protein remains unclear. Recently, the structural and magnetic configurations of the P-cluster oxidation states were shown to coincide with profound differences in the density of low lying electronic states, implying there is a deeper relationship between the electronic-structural properties of the P-cluster and its function in electron transfer.¹⁰

Resolving the relationship between the magnetism and structure of the P-cluster, most notably for the metastable P⁺ state, is important for elucidating a complete mechanistic understanding of the P-cluster in the nitrogenase electron transfer cycle. Herein, we address this goal by combining light-controlled reduction of MoFe protein in complexes with cadmium sulfide nanocrystals (CdS)^16,17 with EPR to resolve magnetic changes in the P-cluster during electron transfer that arise from discrete electronic-structural intermediates in the reduction of P²⁺ to P⁺.

An oxidized sample of nitrogenase MoFe protein was mixed with mercaptopropionic acid capped CdS quantum dots (Figure S1; see Supporting Information for details), and the P-cluster P²⁺ oxidation state was verified by EPR (Figure S2). The CdS:MoFe protein complexes were illuminated with a 405 nm LED at either 231 K or 263 K and then allowed to anneal in the dark at 236 K or 263 K, respectively, to prevent further light-driven reduction. By illuminating at subambient temperatures, the light-driven redox (i.e., electron transfer) process is decoupled from temperature sensitive chemical (i.e., ligand switching) steps during electron transfer and P-cluster conversion from P²⁺↔P⁺. As shown in Figure 2A, illumination at 263 K for 12 min resulted in reduction of the P-cluster exemplified by loss of P²⁺ intensity (36%, Figure S2 and Table S1). This change coincided with the appearance of an S = 1/2 rhombic signal at g = 2.05, 1.94, 1.81 (P⁺_1.81, Figure 2A)^13,15,18 and an S = 1/2 axial signal with g = 2.006, 1.89 (assigned to P⁺_1.89, Figure 2B).^13,14 Dark annealing at 263 K for 20 min (Figure 2A, green trace) led to complete loss of the P⁺_1.89 signal, and an increase in amplitude of the P⁺_1.81 (21%) and P²⁺ (7%) signals (Table S1).

Figure 2.

Illumination and EPR spectra of CdS:MoFe protein complexes at 263 K and 231 K. (A) T = 263 K. Blue trace, oxidized CdS:MoFe protein complexes. Red trace, after 12 min illumination at 263 K. Green trace, spectrum after incubation in the dark at 263 K for 20 min. (B) Illuminated (red trace) minus dark (green trace) difference spectrum. Simulation (black dashed trace) using g = 2.006, 1.89 assigned to the S = 1/2, P⁺_1.89 signal. Buffer pH = 7; EPR conditions, T = 12 K, microwave power = 1 mW. (C) T = 231 K. Blue trace, oxidized CdS:MoFe protein complexes. Red trace, after 15.5 min illumination at 231 K. (D) Low-field EPR spectrum showing the high-spin, S = 7/2, g = 6.54 signal assigned as P⁺_6.54. Sample pH = 7. EPR conditions: (C) T = 12 K, microwave power = 1 mW, (D) T = 18 K, microwave power = 25 mW. Populations of EPR signals are summarized in Table S1.

When illuminated at a lower temperature of 231 K for 15.5 min, reduction of the MoFe protein P-cluster led to a decrease in the P²⁺ signal intensity (19%) and appearance of the P⁺_1.89 signal, whereas formation of the P⁺_1.81 signal was suppressed. Rather, a low-field inflection at g = 6.54 (Figure 2D) appeared, resembling other high-spin signals observed for MoFe protein (Table S2, Figure S3). Rhombogram analysis and simulation of the g = 6.54 signal (referred to as P⁺_6.54) indicates that it originates from a S = 7/2 spin system with E/D ≈ 0.024 (D = −3.2 cm^–1) of the reduced P-cluster, where E and D¹⁹ are the zero-field splitting parameters (see Figure S3 for details).

Dark annealing at 236 K of the CdS:MoFe protein complexes illuminated at 231 K (Figure 2C) was used to monitor relative intensities of P⁺ intermediates following light-driven electron transfer to MoFe protein (Figure S4). EPR spectra of CdS or MoFe protein alone, before and after illumination and annealing, or of CdS:MoFe protein prior to illumination, did not produce any detectable signal changes (Figure S5). Simulations of the low-field regions using singular value decomposition (SVD, Figure S6) and the high-field region using EasySpin²⁰ (Table S3, Figure S7) enabled time-dependent changes in signal intensities of P⁺ intermediates (P⁺_1.89, and P⁺_1.81 and P⁺_6.54) to be fit to reaction models (Tables S4 and S5, Figure S8). The P⁺ signal intensity versus annealing time best fit to a three-step reaction model as summarized in eqs 2 and 3:

2

3

The fit shown in Figure 3 gave relative values for rate constants where k₂ > k₁ > k₃ and predicts the high-spin P⁺_6.54 P-cluster intermediate originates together with P⁺_1.89 under photochemical reduction of the P²⁺ state (Figure 4). In the dark, P⁺_6.54 partitions rapidly between P⁺_1.89 or P⁺_1.81. Therefore, a lack of P⁺_6.54 under illumination at 263 K (Figure 2) is likely due to more rapid conversion to either P⁺_1.81 or P⁺_1.89 (k₁ and k₂ > k₃) than at 231 K.

Figure 3.

Time-dependent changes of the P⁺ EPR signals intensities in CdS:MoFe protein complexes. Initial (time = 0 min) P⁺ signal intensities were collected at 231 K. Changes are plotted versus time under dark annealing at 236 K. P⁺ signal intensities were determined using EasySpin and SVD analysis (see Supporting Information, Figures S6 and S7).²⁰ Solid lines are fits of the experimental data to differential equations; dP⁺_1.89/dt = k₁[P⁺_6.54] – k₃[P⁺_1.89], dP⁺_1.81/dt = k₂[P⁺_6.54] + k₃[P⁺_1.89], and dP⁺_6.54/dt = −(k₁ + k₂)[P⁺_6.54] (Table S4). Green, P⁺_6.54; blue, P⁺_1.89; red, P⁺_1.81.

Figure 4.

Schematic representation of the P²⁺ to P⁺ conversion in low temperature photochemical reduction of the MoFe protein P-cluster. Photoexcitation at 231 K of CdS:MoFe protein complexes poised in P²⁺ (left) leads to electron injection into the P-cluster and reduction to a mixed population of P⁺ states; the S = 7/2 P⁺_6.54 and S = 1/2 P⁺_1.89, which are based on the reaction model (Figure 3), correspond to distinct conformers (inset). Dark annealing at 236 K results in the conversion of P⁺_6.54 to either P⁺_1.89 (faster) or P⁺_1.81 (slower), and conversion of P⁺_1.89 to P⁺_1.81. The rate constants of the conversion of P⁺ states are obtained from fits shown in Figure 3 to a reaction model in Table S4.

Dark annealing was performed over a range of 231 K to 245 K to obtain the temperature-dependence of k₃ for the P⁺_1.89↔P⁺_1.81 step (eq 3, Figure S9, Table S6). An Arrhenius plot of ln k₃ vs 1/T gave a value of E_a = 24 ± 8.3 kcal mol^–1. The value suggests the P⁺_1.89↔P⁺_1.81 involves structural changes in MoFe protein at the P-cluster. For example, reductive formation of P⁺_1.81 from P²⁺ at 298 K is pH-dependent (Figure 1) and is favored at pH ≈ 6 and nearly undetectable at basic pH (>8).¹⁵ Likewise, chemical oxidation of MoFe protein P-cluster from P^N↔P⁺ at 298 K led to formation of both P⁺_1.81 and P⁺_1.89,¹³ with P⁺_1.89 intensity being maximal at pH 8.4.¹⁴ The two results are consistent with formation of P⁺_1.89 being reversible and both pH- and temperature-dependent.

In addition to analysis of the P⁺_1.89 intermediate, photochemical reduction of MoFe protein at 231 K also enabled assignment of the P⁺_6.54 high-spin state to a unique electron transfer intermediate (Figure 3). In the P²⁺↔P⁺ reduction step, the high-spin P⁺_6.54 state has two possible fates: direct conversion to P⁺_1.81 (eq 3) where k₂ > k₃ or rapid conversion to P⁺_1.89 followed by slow P⁺_1.89↔P⁺_1.81 conversion (eq 2). Thus, the reaction model for the P-cluster P²⁺↔P⁺ conversion, summarized in Figure 4, involves two spin-state isomer intermediates. The observation of multiple electronic intermediates associated with a redox step in the P-cluster is similar to the observation of low-spin and high-spin S₂ states of the PSII oxygen evolving complex that arise from valence isomerism in Mn–O–Mn coordination from different S₂ conformers that function in the catalytic cycle of water oxidation.²¹⁻²⁵ The interconversion of P⁺_6.54↔P⁺_1.89 may likewise arise from conformational isomerism in Fe-coordination to S₁ (see Figure 1) that guide formation of P⁺ with surrounding structural changes. Overall, the results from combining low temperature photochemical reduction of the MoFe protein with dark annealing reveal that formation of the metastable P⁺ state, P⁺_1.81, involves intermediate spin states and electronic configurations that occur with changes in P-cluster coordination.

As established by kinetic and theoretical studies, correlated motions within the nitrogenase complex during turnover have an important function in enabling P-cluster mediated electron transfer to be integrated with catalysis.^7−9,26 As shown here, during electron transfer, there are also discrete changes in P-cluster magnetic structure that are linked to changes in oxidation state. The EPR analysis and kinetic model are most consistent with these magnetic states originating from different P-cluster conformers during electron transfer and reduction of P²⁺ to P⁺, which may function in the electron transfer mechanism within the nitrogenase complex during ammonia production.²⁷

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.1c13311.

• A description of experimental procedures including nanocrystal synthesis, EPR spectroscopy and simulation, and reaction models. Figures of parallel mode and low-field perpendicular mode EPR, a detailed analysis for the high-field P⁺_6.54 signal assignment, and temperature-dependence analysis of P⁺_1.89↔P⁺_1.81 EPR signals; Tables of spectral analysis and reaction model fits (PDF).

The authors declare no competing financial interest.