A VTVH MCD and EPR Spectroscopic Study of the Maturation of the “Second” Nitrogenase P-Cluster

Abstract

The P-cluster of the nitrogenase MoFe protein is a [Fe₈S₇] cluster that mediates efficient transfer of electrons to the active site for substrate reduction. Arguably the most complex homometallic FeS cluster found in nature, the biosynthetic mechanism of the P-cluster is of considerable theoretical and synthetic interest to chemists and biochemists alike. Previous studies have revealed a biphasic assembly mechanism of the two P-clusters in the MoFe protein upon incubation with Fe protein and ATP, in which the first P-cluster is formed through fast fusion of a pair of [Fe₄S₄]⁺ clusters within 5 min and the second P-cluster is formed through slow fusion of the second pair of [Fe₄S₄]⁺ clusters in a period of 2 h. Here we report a VTVH MCD and EPR spectroscopic study of the biosynthesis of the slow-forming, second P-cluster within the MoFe protein. Our results show that the first major step in the formation of the second P-cluster is the conversion of one of the precursor [Fe₄S₄]⁺ clusters into an integer spin cluster [Fe₄S_3-4]^α, a process aided by the assembly protein NifZ; whereas the second major biosynthetic step appears to be the formation of a diamagnetic cluster with a possible structure of [Fe₈S_7-8]^β, which is eventually converted into the P-cluster.

Graphical abstract

An Electron Paramagnetic Resonance/Magnetic Circular Dichroism spectroscopic study of the maturation of the two [Fe₄S₄]⁺ precursor clusters on the nitrogenase MoFe protein (NifDK) into the second P-cluster (Fe₈S₇) reveals that the first step of the reaction is the conversion of one of the [Fe₄S₄]⁺ clusters into an integer spin cluster, possibly the all-ferrous cluster [Fe₄S₄]⁰.

Nitrogen fixation, the biological conversion of dinitrogen to ammonia, is catalyzed by the enzyme nitrogenase¹. The best characterized form of this enzyme, Mo-nitrognease, consists of two separable proteins, the Fe protein (encoded by nifH) and the MoFe protein (encoded by nifDK), both of which are required for the enzymatic activity of nitrogenase. The Fe protein is a γ₂ homodimer containing one MgATP binding site within each subunit and a subunit-bridging [Fe₄S₄] cluster². The MoFe protein is an α₂β₂ tetramer housing two pairs of metal clusters: the P-cluster and the FeMo-cofactor (or FeMoco). The P-cluster is a [Fe₈S₇] unit bridging each αβ dimer (Figure 1A, B), whereas the FeMoco is a [MoFe₇S₉C-homocitrate] cluster located within each α subunit^3–5. During catalysis, electrons are transferred from the P-cluster to the FeMoco of the MoFe protein⁶, where substrate reduction takes place followed by transfer from the [Fe₄S₄] cluster of the Fe protein to the P-cluster concomitant with ATP hydrolysis.

Figure 1.

Structures of the P-cluster in the reduced, P^N (A) and oxidized, P²⁺ (B) states and the P-cluster precursor in ΔnifH and ΔnifBZ MoFe proteins (C). The structures shown in A and B are crystallographic observations⁷ and the structure shown in C is an XAS/EXAFS-derived model⁸.

The assembly of P-cluster, the “capacitor” mediating the electron transfer to the FeMoco during catalysis, has attracted considerable attention^9–10. The precursor of the P-cluster was identified through the characterization of a FeMoco-deficient MoFe protein (designated ΔnifH MoFe protein)^7–11 generated in vivo upon deletion of nifH, the gene encoding the Fe protein that is essential for FeMoco maturation. The ΔnifH MoFe protein contains two pairs of neighboring [Fe₄S₄]-like clusters (Figure 1C) at the equivalent positions of the two P-clusters^7–11. In Nature, these clusters can exist in four different paramagnetic states, [Fe₄S₄]⁰ (S = 4), [Fe₄S₄]⁺ (S = ½, $\raisebox{1ex}{$3$}\!\left/ \!\raisebox{-1ex}{$2$}\right.,\mathrm{ }\raisebox{1ex}{$5$}\!\left/ \!\raisebox{-1ex}{$2$}\right.$), [Fe₄S₄]²⁺ (S ≥ 1 or 0) and [Fe₄S₄]³⁺ (S = ½)^12–14. Most surprisingly, chemical oxidation of the all-ferrous clusters can convert the precursors into P-clusters in a non-enzymatic reaction¹⁴.

The biosynthesis of the P-cluster can be studied through incubation of ΔnifH MoFe protein with Fe protein, ATP, and reductant^{11, 15}. The time-dependent profile of P-cluster biosynthesis in ΔnifH MoFe protein reveals that this process occurs in two phases: an initial fast phase (0-5 min), during which one P-cluster is synthesized; and a second slow phase (ca. 2 h), during which the second P-cluster is formed^15–16. To date, details of the mechanism for the formation of either cluster are unknown.

The assembly of the second P-cluster can be further investigated through the characterization of another FeMoco-deficient MoFe protein (designated ΔnifBZ MoFe protein), which is generated in vivo upon deletion of nifB and nifZ, the genes encoding proteins essential for the synthesis of the FeMoco and the second P-cluster, respectively^17–18. The ΔnifBZ MoFe protein contains one complete P-cluster and a [Fe₄S₄] cluster pair, like that found in the ΔnifH MoFe protein, at the second P-cluster site¹⁷. The biosynthesis of the second P-cluster can be achieved through incubation of the ΔnifBZ MoFe protein with NifZ, followed by incubation of this protein with Fe protein, ATP, and reductant. The time-dependent profile of the P-cluster synthesis in ΔnifBZ MoFe protein is essentially identical to that of the second P-cluster in ΔnifH MoFe protein, suggesting that both reactions follow the same mechanism¹¹.

In this work, we use variable-temperature, variable-field magnetic circular dichroism (VTVH MCD) and EPR spectroscopies to further explore the formation of the second P-cluster in both ΔnifBZ and ΔnifH MoFe proteins. Our results show that the formation of the second P-cluster undergoes an initial conversion of one of the precursor [Fe₄S₄]⁺ clusters into an integer spin cluster [Fe₄S_3-4]^α in a process facilitated by NifZ, followed by possible formation of a diamagnetic cluster with a proposed structure [Fe₈S_7-8]^β prior to conversion of this intermediate into a mature P-cluster.

Results

Biosynthesis of the second P-cluster in ΔnifBZ MoFe protein.

The MCD spectrum of the reduced ΔnifBZ MoFe protein has been published previously¹⁷ and shown to exhibit the classic inflections and amplitude of two [Fe₄S₄]⁺ clusters. The magnetization curves of ΔnifBZ MoFe protein can be simulated by adding ½ of the spectrum of reduced ΔnifH MoFe protein (representing two [Fe₄S₄]⁺ clusters) and ½ of the spectrum of reduced ΔnifB MoFe protein (representing one diamagnetic P-cluster, designated P^N), consistent with the characterization of a MoFe protein containing two [Fe₄S₄]⁺ clusters and one complete P-cluster.

The MCD spectrum of oxidized ΔnifBZ MoFe protein (Figure 2), on the other hand, has not been reported so far. Like the spectrum of the reduced protein, the spectrum of the oxidized protein can be simulated by adding ½ of the spectrum of the oxidized ΔnifH MoFe protein (representing two paramagnetic [Fe₄S₄]²⁺ clusters) and ½ of the spectrum of the oxidized ΔnifB MoFe protein (representing one paramagnetic oxidized P-cluster, designated P²⁺). Therefore, the simulations of both reduced and oxidized proteins clearly verify that ΔnifBZ MoFe protein contains one complete P-cluster and two neighboring [Fe₄S₄] clusters that are electronically and structurally identical to those in the precursor ΔnifH MoFe protein.

Figure 2.

Comparison between the MCD spectrum of IDS-oxidized ΔnifBZ MoFe protein (red) and a simulated MCD spectrum (blue; 80 mg/mL) generated by adding ½ of the spectrum of IDS-oxidized ΔnifB MoFe protein (representing one P²⁺ signal; 14.5 mg/mL) and ½ of the spectrum of IDS-oxidized ΔnifH MoFe protein (representing two paramagnetic [Fe₄S₄]²⁺ clusters; 34 mg/mL). The strong similarity between the two spectra shows that the [Fe₄S₄]²⁺ clusters in ΔnifBZ MoFe protein remain paramagnetic as they are in ΔnifH MoFe protein. Spectra were recorded at 1.6 K and 6.0 T.

Figure 3 shows the MCD spectrum of the reduced ΔnifBZ MoFe protein following incubation with NifZ (designated ΔnifBZ MoFe protein^+NifZ). The electronic structure of the cluster species in ΔnifBZ MoFe protein^+NifZ clearly arises from a [Fe₄S₄]⁺ cluster¹⁹, with its inflections in the MCD spectrum being essentially identical to those in both ΔnifBZ and ΔnifH MoFe proteins. However, unlike ΔnifBZ and ΔnifH MoFe proteins that exhibit distinct S = ½ EPR signals, the ΔnifBZ MoFe protein^+NifZ is practically EPR silent (Figure 4). These remaining signals consist of a small radical-like g 2 signal and a rolling background distinctly different from the original ΔnifBZ signal. The source of these signals is unknown.

Figure 3.

MCD spectra of dithionite-reduced ΔnifH MoFe protein (blue; 70 mg/mL), ΔnifBZ MoFe protein (red; 40 mg/mL) and ΔnifBZ MoFe protein^+NifZ (green; 76 mg/mL). Spectra were recorded at 1.6 K and 6.0 T.

Figure 4.

EPR spectra of dithionite-reduced ΔnifBZ MoFe protein (red) and ΔnifBZ MoFe protein^+NifZ (blue). Spectra were recorded at 9.48 GHz, 10 K, 25 mW microwave power and modulation amplitude of 0.5 mT.

A comparison between the magnetization curves of ΔnifBZ MoFe protein with those of ΔnifBZ MoFe protein^+NifZ (Figure 5) reveals that, while the curves of ΔnifBZ MoFe protein exhibit the general shape associated with the S = ½, [Fe₄S₄]⁺ clusters with a small linear diamagnetic contribution due to the presence of the P-cluster (S = 0)¹⁷, the curves of ΔnifBZ MoFe protein^+NifZ exhibit complex fluctuations with both temperature and magnetic field. These fluctuations suggest the presence of a second paramagnetic species, different from the normal S = ½ [Fe₄S₄]⁺ cluster, in ΔnifBZ MoFe protein^+NifZ.

Figure 5.

MCD magnetization curves (plotted versus B rather than the usual βB/kT) of dithionite-reduced ΔnifBZ MoFe protein before (A) and after (B) incubation with NifZ. The magnetization curves of ΔnifBZ MoFe protein (A) represent the sum of the paramagnetic contribution (C term) of the [Fe₄S₄]⁺ clusters and the linear diamagnetic contribution (B term) of the P^N cluster. Upon incubation with NifZ, the magnetization curves of ΔnifBZ MoFe protein^+NifZ (B) show a distinct curvature at higher temperatures. The magnetization curves were recorded at 520 nm, 6 T and 1.6 K (blue), 4.2 K (red) and 10 K (green), respectively.

The MCD spectrum of the oxidized ΔnifBZ MoFe protein^+NifZ (Figure 6) shows both similarities and differences when compared with that of the oxidized ΔnifBZ MoFe protein. On the one hand, both proteins are similar in that they exhibit a strong spectral inflection at 800 nm that is associated with a single oxidized P-cluster (P²⁺). On the other hand, the inflections associated with the two paramagnetic [Fe₄S₄]²⁺ clusters in ΔnifBZ MoFe protein are absent from the spectrum of the oxidized ΔnifBZ MoFe protein^+NifZ.

Figure 6.

Comparison between the MCD spectrum of IDS oxidized IDS-oxidized ΔnifBZ MoFe protein^+NifZ (green; 71 mg/mL) and the simulated spectrum (blue) generated by adding ½ of the spectrum of IDS-oxidized ΔnifB MoFe protein (representing one P²⁺ signal; 14.5 mg/mL) and ¼ of the spectrum of IDS-oxidized ΔnifH MoFe protein (representing one paramagnetic [Fe₄S₄]²⁺ cluster; 34 mg/mL). The lack of structure associated with the paramagnetic [Fe₄S₄]²⁺ cluster (see Figure 2) in the spectrum of ΔnifBZ MoFe protein^+NifZ shows that, like all other classic [Fe₄S₄]²⁺ clusters, the [Fe₄S₄]²⁺ cluster in ΔnifBZ MoFe protein^+NifZ is now diamagnetic. Spectra were recorded at 1.6 K and 6.0 T.

Biosynthesis of the second P-cluster in ΔnifH MoFe protein.

The biphasic formation of the two P-clusters in ΔnifH MoFe protein was monitored by EPR and MCD spectroscopic techniques. Consistent with the formation of P-clusters, the MCD spectrum of the oxidized ΔnifH MoFe protein (Figure 7) exhibits a broad transition at 800 nm (mainly due to P²⁺) that increases in intensity with the incubation time of ΔnifH MoFe protein with Fe protein, ATP and reductant. Parallel mode EPR spectroscopy of ΔnifH MoFe protein¹⁶ shows a similar increase in intensity of the signal at g = 11.9 with incubation time, which is characteristic of the P²⁺ state of the P-cluster.

Figure 7.

MCD spectra recorded during the biosynthesis of the two P-clusters in the IDS-oxidized ΔnifH MoFe protein at 5 min (blue; 40.1 mg/mL), 20 min (red; 58mmg/mL) and 60 min (green; 79.3 mg/mL) in comparison with the MCD spectrum of ΔnifB MoFe protein (black; 56 mg/mL) that contains two P-clusters. Note that the intensity of the P²⁺ clusters seen at 800 nm increases with time as the P-clusters are being synthesized. During the synthesis of the P-cluster, inflections arise in the 400–600 nm region, which are not associated with the P²⁺ cluster, but possibly associated with the predicted intermediate(s) A and/or B. Spectra were recorded at 1.6 K and 6.0 T.

A similar study of reduced ΔnifH MoFe protein reveals even more interesting details. Magnetization curves recorded at 0 min (Figure 8) exhibit shapes characteristic of predominately an S = ½ system, consistent with the EPR spectrum and previous MCD spectroscopic studies¹². At 5 and 20 min of incubation, however, the magnetization curves exhibit strong deviation from a simple S = ½ system, similar to the curves obtained for ΔnifBZ MoFe protein^+NifZ (Figure 5).

Figure 8.

MCD magnetization curves (plotted versus B rather than the usual βB/kT) recorded during the biosynthesis of the two P-clusters in the dithionite-reduced ΔnifH MoFe protein at 0 min (A), 5 min (B) and 20 min (C). Spectra were recorded at 520 nm, 6.0 T and 1.6 K (blue), 4.2 K (red) and 10 K (green), respectively. Note that the curves at 1.6 K closely mimic the curves of a standard S = ½ spin system, whereas the curves at 4.2 K and 10 K show strong deviations from an S = ½ system, suggesting the presence of another spin system that is likely integer.

Figure 9 shows the MCD spectrum of ΔnifH MoFe protein after 5 min of incubation, when approximately one pair of [Fe₄S₄] clusters is converted into a P-cluster and the second pair of [Fe₄S₄] clusters remains in the precursor state. Interestingly, the MCD spectrum exhibits inflections characteristic of [Fe₄S₄]⁺ clusters with a spectral intensity suggesting ca. 1.4 clusters per protein; whereas the EPR spectrum associated with the [Fe₄S₄]⁺ clusters is much smaller, corresponding to ca. 0.3 clusters per protein. While the MCD spectral amplitudes correlate with the concentration of [Fe₄S₄]⁺ clusters, EPR spectral intensities can be diminished (e.g., broadened) by the presence of strong dipolar and exchange interactions. Similar interactions may also be present in ΔnifBZ MoFe protein^+NifZ and may account for the lack of a significant EPR spectrum in that protein. After 20 min of incubation, the MCD spectral intensity of the [Fe₄S₄]⁺ clusters in ΔnifH MoFe protein continues to decrease as more P-clusters are formed. Surprisingly, the MCD signal intensity increases again at 60 min of incubation with a further increase at 120 min.

Figure 9.

MCD spectra recorded during the biosynthesis of the two P-clusters in the dithionite-reduced ΔnifH MoFe protein at 0 min (black; 113.2 mg/mL), 5 min (red), 20 min (blue), 60 min (green) and 120 min (brown). Note that the intensity of the [Fe₄S₄]⁺-like spectrum decreases in the first 20 min but increases afterwards to ca. 2 clusters per protein. Spectra were recorded at 1.6 K and 6.0 T.

Discussion

Effect of incubating ΔnifBZ MoFe protein with NifZ.

NifZ is a small (19 kDa), non-redox-active protein that has been shown to be essential for the biosynthesis of the second, slow-forming P-cluster. However, while NifZ must interact with ΔnifBZ MoFe protein to enable the formation of the second P-cluster, it does not have to be present during the subsequent coupling of two [Fe₄S₄] clusters into a mature P-cluster.

The MCD spectrum of ΔnifBZ MoFe protein^+NifZ exhibits inflections similar to those of both ΔnifH and ΔnifBZ MoFe proteins (Figure 3). What is different among these spectra is their intensity. The spectral intensity at 520 nm of a typical [Fe₄S₄]⁺ cluster^20–21 is 60–90 Δε M¹⁻ cm¹⁻. Using that parameter, the intensity of the ΔnifH MoFe protein spectrum correlates to four [Fe₄S₄]⁺ clusters, consistent with the previous biochemical and spectroscopic analysis of this protein. Likewise, consistent with the presence of only two [Fe₄S₄]⁺ clusters in ΔnifBZ MoFe protein, the spectral intensity of this protein is approximately half (~46%) of that of ΔnifH MoFe protein. Unexpectedly, the spectral intensity of ΔnifBZ MoFe protein^+NifZ is only one fourth (~27%) of that of ΔnifH MoFe protein, suggesting the presence of only one [Fe₄S₄]⁺ cluster rather than the expected two in this protein.

What happened to the second [Fe₄S₄]⁺ cluster? The complexity of the magnetization curves of ΔnifBZ MoFe protein^+NifZ, when compared to those of the ΔnifBZ MoFe protein, shows that the cluster species in ΔnifBZ MoFe protein^+NifZ is not a pure S = ½ system and suggests the presence of a second high-spin paramagnetic cluster. Additionally, ΔnifBZ MoFe protein^+NifZ is essentially EPR-silent, further suggesting that the second cluster became high-spin paramagnetic, which would likely induce spectral broadening through dipolar and/or exchange interactions.

The logical conclusion is that the second cluster is an integer spin (non-Kramer) state. But, if the second cluster is paramagnetic, why does it contribute little, if any, intensity to the MCD spectrum at 1.6 K? One answer to that question is that the second cluster could be an integer spin state with a near axial (E ≈ 0) symmetry and positive zero-field splitting, D > 0. In that situation, the ground state would be non-degenerate (m_s = 0) and would, therefore, contribute almost no MCD spectral intensity¹⁹ at 1.6 K. As the temperature rises, degenerate excited states (e.g., m_s = ±1) would become populated and contribute to both the MCD spectrum and magnetization curves. This is consistent with the data showing that the magnetization curve of the ΔnifBZ MoFe protein^+NifZ at 1.6 K nearly mimics an S = ½ system but deviates dramatically at higher temperatures (Figure 5).

Another possibility is that the second cluster has become an all-ferrous, [Fe₄S₄]⁰ cluster. The [Fe₄S₄]⁰ cluster has been previously shown to be an intermediate in the non-enzymatic synthesis of the P-cluster¹⁴ and, as such, may also be an intermediate in the biosynthesis of this cluster. The ground state of [Fe₄S₄]⁰ is typically paramagnetic with S = 4 (D < 0), as verified by EPR and Mössbauer spectroscopic studies. The possibility that the second cluster is [Fe₄S₄]⁰ can be easily tested through spectral simulation. The MCD spectrum of the dithionite-reduced ΔnifBZ MoFe protein corresponds to two [Fe₄S₄]⁺ clusters, while the spectrum of the Ti(III) citrate reduced ΔnifH MoFe protein corresponds to four [Fe₄S₄]⁰ clusters¹⁴. Adding ½ the spectrum of the dithionite-reduced ΔnifBZ MoFe protein and ¼ of the spectrum of Ti(III) citrate-reduced protein should closely simulate the spectrum of ΔnifBZ MoFe protein^+NifZ, assuming the protein in question contains one [Fe₄S₄]⁺ cluster and one [Fe₄S₄]⁰ cluster.

Figure 10 shows the simulated spectrum at 1.6 K, which yields a very good fit to the experimental spectrum of ΔnifBZ MoFe protein^+NifZ. The reason for this close fit is easy to explain. The transitions in the MCD spectrum of [Fe₄S₄]⁰ are similar to those of [Fe₄S₄]⁺ but with much lower intensity (all of the iron in [Fe₄S₄]⁰ are ferrous, thus reducing the presence of the normally dominant S-to-Fe charge-transfer bands in the spectrum). Therefore, adding the two spectra yields a spectrum only slightly larger than that of a single [Fe₄S₄]⁺ cluster. As the temperature increases, the relative spectral contribution of the high-spin S = 4 [Fe₄S₄]⁰ cluster will increase compared to that of the low-spin S = ½ [Fe₄S₄]⁺ cluster, resulting in magnetization curves that greatly deviate from an S = ½ system at higher temperatures (Figure 8).

Figure 10.

Comparison between the MCD spectrum of dithionite-reduced ΔnifBZ MoFe protein^+NifZ (blue) and a simulated spectrum (red) generated by adding ½ the spectrum of the dithionite-reduced ΔnifBZ MoFe protein (representing one [Fe₄S₄]⁺ cluster) and ¼ the spectrum of the Ti(III) citrate-reduced ΔnifH MoFe protein (representing one S = 4 [Fe₄S₄]⁰ cluster).

Biosynthesis of the second P-cluster in ΔnifH MoFe protein.

The overall synthesis reaction is the conversion of two neighboring [Fe₄S₄]⁺ clusters (collectively abbreviated as P*) in ΔnifH MoFe protein into the P-cluster (abbreviated as P). Three basic mechanisms can be proposed for the synthesis of the second P-cluster: No Intermediates

$${\mathit{P}}^{\ast }\stackrel{k_{01}}{\to }\mathit{P}$$ (1)

One Intermediate

$${\mathit{P}}^{\ast }\stackrel{k_{11}}{\to }A\stackrel{k_{12}}{\to }\mathit{P}$$ (2)

Two Intermediates

$${\mathit{P}}^{\ast }\stackrel{k_{21}}{\to }\mathit{A}\stackrel{k_{22}}{\to }\mathit{B}\stackrel{k_{23}}{\to }\mathit{P}$$ (3)

Best-fit simulations of the experimental data were performed (see Supporting Information) for each mechanism.

It is obvious that the experimental data do not fit the simulation (Figure 11) for Equation (1). Equations (2) and (3) provide better fits of the data, with Equation (3) showing the best fit of the three. For the sake of simplicity and discussion only, it will be assumed that the reaction mechanism has two intermediates (Equation 3). However, mechanisms with more intermediates cannot be excluded.

Figure 11.

Best-fit theoretical predictions of the time-dependent concentration increase of the second P-cluster, assuming no intermediate (Eq. SE1; red), one intermediate (Eq. SE14; green) or two intermediates (Eq. SE15; blue). The average data (blue squares) from EPR spectroscopic studies and activity measurements are also shown in the figure.

As discussed above, MCD spectroscopy is an excellent technique for monitoring the concentration of [Fe₄S₄]⁺ clusters. At 5 min of synthesis the protein should contain approximately one P-cluster, which is diamagnetic and, therefore contribute only slightly to the MCD spectrum¹². This means that the MCD spectra at 5, 20, 60 and 120 min mainly reflect paramagnetic intermediates in the synthesis of the second P-cluster. The two-intermediate kinetic scheme (Equation 3) predicts (Figure S1) that components P* and A will dominate the MCD spectrum at 5 min. The magnetization curves at the 5 min synthesis mark (Figure 8) exhibit fluctuations similar to those of ΔnifBZ MoFe protein^+NifZ (Figure 5), suggesting that both proteins contain similar paramagnetic clusters. These fluctuations are even more pronounced at 20 min (Figure 8). This suggests that the first major step of P* to A in the reaction mechanism for the biosynthesis of the second P-cluster is likely the conversion of one [Fe₄S₄] cluster into an integer spin cluster, [Fe₄S_3–4]^α.

One of the most interesting features of the data collected on reduced ΔnifH MoFe protein is the increase in the MCD spectral concentration during the later stages of P-cluster biosynthesis. Specifically, the initial [Fe₄S₄]⁺-like signal intensity decreases during the first 20 min but then increases with time. Obviously, the increase of the signal intensity cannot correspond to the original [Fe₄S₄]⁺ clusters since those clusters have been converted into P-clusters by 120 min; rather, such an increase must originate from some other FeS cluster species.

Since there are no other proteins present, it is possible that the signal originates from the newly formed second P-cluster. It has been shown that the one-equivalent oxidized P-cluster (P⁺) yields MCD and EPR spectra characteristic of [Fe₄S₄]⁺-like clusters²². Therefore, if the second P-cluster forms as P⁺ rather than P^N, the intensity of the new [Fe₄S₄]⁺-like signal will correlate with the concentration of the second P-cluster. This correlation is demonstrated using the theoretical kinetic equations (i.e., Equations SE7, SE12, SE13 and SE 14) to predict the variation in the intensity of the total [Fe₄S₄]⁺-like MCD signals with time (see Supporting Information).

Figure 12 plots the MCD data (amplitude at 520 nm) along with the predicted MCD spectral intensity using the assumption that the new [Fe₄S₄]⁺-like signal correlates with the formation of the second P-cluster (see Supporting Information).

Figure 12.

Theoretical plot (solid line) of the MCD signal of the reduced ΔnifH MoFe protein versus time (using Eqs. SE7, SE11, SE12 and SE13) assuming the new [Fe₄S₄]⁺-like spectrum is formed in parallel with the synthesis of the second P-cluster. Amplitudes (at 520 nm) of experimental MCD spectra are shown as black circles.

The final question is the identity of intermediate B (Eq. 4). Because B is generated in the middle of the synthesis, at a time point when P*, A, B and P are all present, it is much more difficult to isolate and characterize B. The theoretical reaction profile (Figure S2) suggests that B reaches its maximum intensity between the 20 min and 60 min synthesis marks. However, during that time period, no new inflections are observed in the MCD spectrum of the reduced protein, which suggests that B may be diamagnetic. The MCD spectra of the oxidized system, on the other hand, exhibit new inflections (Figure 9) in the 400–600 nm region, which are different from those associated with pure P²⁺. The perpendicular mode EPR spectrum (Figure S3) of the oxidized system also shows inflections in the g 2 region that grow in intensity with time, maximizing at 60 min yet barely detectable at 120 min. The MCD and EPR spectral inflections may correspond to B⁺ or some other intermediate generated later in the biosynthesis of the P-cluster. Unfortunately, neither the EPR nor the MCD spectrum allows identification of the structure of B. However, it could be proposed that B represents a fusion of the A clusters into a structure ([Fe₈S_7-8]^β) similar to the P-cluster.

Summary

Simulations of the biosynthetic reaction profile of the second P-cluster suggest the appearance of at least two major intermediates during the formation of this P-cluster. One of the initial intermediates (A) appears to involve the conversion of one of the precursor [Fe₄S₄]⁺ clusters into an integer spin cluster, possibly with the loss of an S atom (i.e., [Fe₄S₃]^α). The mechanism for the removal of the surplus sulfide is unclear, although it can be speculated that this sulfide is removed as H₂S or a protein-bound persulfide. The identity of the new cluster is likely either an integer spin state with near axial symmetry (E ~ 0 and D > 0) or the all-ferrous, [Fe₄S₄]⁰ cluster. The current MCD and EPR spectroscopic data are unable to definitively favor one or the other of these two proposed structures for the new cluster. Another major intermediate (B) is more difficult to characterize. The lack of either an obvious MCD or EPR spectrum suggests it as a diamagnetic multinuclear cluster, possibly representing a fusion of the two original clusters into an 8Fe precursor of the eventual P-cluster.

Questions still remain as to why the synthesis of only the second P-cluster requires NifZ and why the formation kinetics of the two P-clusters are so different. It is unlikely that the formation kinetics of the second P-cluster is significantly slower that the first P-cluster in vivo. The in vivo P-cluster maturation could be facilitated by the physiological electron donor(s), which deliver electrons more efficiently to the Fe protein and/or render the Fe protein in an appropriate redox state/conformation for the subsequent reductive coupling of the 4Fe precursors into a P-cluster. A similar discrepancy between the in vivo and in vitro activities has been observed in the case of CO₂ reduction to CO by A. vinelandii Fe proteins, where the turnover number of the in vitro assay (where dithionite is supplied as an electron source) is considerably lower than that under the in vivo conditions²³. Moreover, chaperon proteins, such as GroEL, have been implicated in the nitrogenase maturation process²⁴. Therefore, the possible participation of chaperons and other accessory proteins in this process in vivo cannot be excluded. While NifZ has been proposed to act as a clamp that prepares the site of the second P-cluster for maturation, the implication of the two introductory questions suggests a cooperative interaction between the clusters even though they are ca. 60 Å apart and thereby provide a basic framework for further investigations into the mechanistic details of P-cluster biosynthesis in the future.