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

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, PN, P+, P2+ of which PN↔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.

N itrogenase 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 N 2 reduction to ammonia according to eq 1: 1 During turnover, the electrons required for N 2 reduction are transferred from Fe protein to MoFe protein, which is an α 2 β 2 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 N 2 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 II Fe 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 P 2+ ([6Fe II 2Fe III -7S] +2 ) ( Figure 1). 5 Transitions between Pcluster states involve extensive structural changes, including a switch in Fe-coordination of the central sulfide (S 1 ) that bridges the two [4Fe-3S] subclusters, and amide nitrogen coordination to Fe 5 by α-88 cysteine (α-88Cys) and β-188 serine (β-188Ser) oxygen coordination to Fe 6 . Recently, the Xray structure of MoFe protein was solved with the P-cluster poised in the P + state, with an intermediate structural arrangement between P 2+ and P N (Figure 1). 6 In the P + state, the S 1 sulfide is pentacoordinate and β-188Ser coordinates Fe 6 . The observation of structural changes in the MoFe protein P-cluster has been incorporated into conformational gating 7 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 The structural rearrangements of the P-cluster in different oxidation states also coincide with changes in spin states and EPR properties. P 2+ 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 electronicstructural 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 lightcontrolled 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 P 2+ 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 P 2+ 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 P 2+ ↔P + . As shown in Figure 2A, illumination at 263 K for 12 min resulted in reduction of the P-cluster exemplified by loss of P 2+ 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 P 2+ (7%) signals (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 P 2+ 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 D 19 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 lightdriven 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 EasySpin 20 (Table S3, Figure S7) enabled timedependent 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   Table S1. annealing time best fit to a three-step reaction model as summarized in eqs 2 and 3: The fit shown in Figure 3 gave relative values for rate constants where k 2 > k 1 > k 3 and predicts the high-spin P + 6.54 P-cluster intermediate originates together with P + 1.89 under photochemical reduction of the P 2+ 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 (k 1 and k 2 > k 3 ) than at 231 K. Dark annealing was performed over a range of 231 K to 245 K to obtain the temperature-dependence of k 3 for the P + 1.89 ↔ P + 1.81 step (eq 3, Figure S9, Table S6). An Arrhenius plot of ln k 3 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 P 2+ 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 P 2+ ↔P + reduction step, the high-spin P + 6.54 state has two possible fates: direct conversion to P + 1.81 (eq 3) where k 2 > k 3 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 P 2+ ↔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 S 2 states of the PSII oxygen evolving complex that arise from valence isomerism in Mn−O−Mn coordination from different S 2 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 S 1 (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][8][9]26 As shown here, during electron transfer, there are also discrete changes in Pcluster 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 P 2+ to P + , which may function in the electron transfer mechanism within the nitrogenase complex during ammonia production. 27 ■ ASSOCIATED CONTENT
A description of experimental procedures including nanocrystal synthesis, EPR spectroscopy and simulation, and reaction models. Figures of parallel mode and lowfield perpendicular mode EPR, a detailed analysis for the high-field P + 6.54 signal assignment, and temperature-  Figures S6 and S7). 20 Solid lines are fits of the experimental data to differential equations; dP + 1.89 /dt = k 1 [P + 6.54 ] − k 3 [P + 1.89 ], dP + 1.81 /dt = k 2 [P + 6.54 ] + k 3 [P + 1.89 ], and dP + 6.54 /dt = −(k 1 + k 2 )[P + 6.54 ] (Table S4). Green, P + 6.54 ; blue, P + 1.89 ; red, P + 1.81 . Figure 4. Schematic representation of the P 2+ 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 P 2+ (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. dependence analysis of P + 1.89 ↔P + 1.81 EPR signals; Tables of spectral analysis and reaction model fits (PDF).