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. 2022 Feb 11;12(9):5214–5224. doi: 10.1039/d1ra08507g

Statistical analysis of PN clusters in Mo/VFe protein crystals using a bond valence method toward their electronic structures†

Chang Yuan 1, Wan-Ting Jin 1, Zhao-Hui Zhou 1,✉
PMCID: PMC8981338  PMID: 35425536

Abstract

Nowadays, large numbers of MoFe proteins have been reported and their crystal data obtained by X-ray crystallography and uploaded to the Protein Data Bank (PDB). By big data analysis using a bond valence method, we make conclusions based on 79 selected PN in all 119 P-clusters of 53 MoFe proteins and 10 P-clusters of 5 VFe proteins from all deposited crystallographic data of the PDB. In the condition of MoFe protein crystals, the resting state PN clusters are proposed to have the formal oxidation state of 2Fe(iii)6Fe(ii), hiding two oxidized electron holes with high electron delocalization. The calculations show that Fe1, Fe2, Fe5, Fe6 and Fe7 perform unequivocally as Fe2+, and Fe3 is remarkably prone to Fe(iii), while Fe4 and Fe8 have different degrees of mixed valences. For PN clusters in VFe protein crystals, Fe1, Fe2, Fe4, Fe5 and Fe6 tend to be Fe2+, but the electron distributions rearrange with Fe7 and Fe8 being more oxidized mixed valences, and Fe3 presenting a little more reductive mixed valence than that in MoFe proteins. In terms of spatial location, Fe3 and Fe6 in P-clusters of MoFe proteins are calculated as the most oxidized and reduced irons, which have the shortest distances from homocitrate in the FeMo-cofactor and [Fe4S4] cluster, respectively, and thus could function as potential electron transport sites. This work shows different electron distributions of PN clusters in Mo/VFe protein crystals, from those obtained from previous data from solution with excess reducing agent from which it was concluded that PN clusters are all ferrous according to Mössbauer and electron paramagnetic resonance spectra.

Iron valences of 129 P-clusters from FeMo/V proteins were analyzed using a bond valence method, supposing the existence of Fe3+ in a generally considered all-ferrous PN cluster in solution with excess reducing agent.graphic file with name d1ra08507g-ga.jpg

1. Introduction

Nitrogenase is a biological enzyme that can activate the triple bond of N2 to form ammonia at moderate temperature and pressure, as shown in eqn (1) below.

N2 + 8e− + 8H+ + 16ATP → 2NH3 + H2 + 16ADP + 16Pi 1

Given that over half of the fixed N inputs that sustain the earth's population are supplied biologically,1 it is necessary to understand the mechanism of N2 fixation. In nitrogenase, three metalloclusters participate in the catalytic process: [Fe4S4] designated as the F-cluster, Mo*Fe7S9C(R-Hhomocit*) (H4homocit = homocitric acid, Hcys = cysteine, Hhis = histidine)2–6 referred to as the FeMo-cofactor (FeMo-co) or M-cluster, and [Fe8S7] named the P-cluster.7,8 In an iron protein, [Fe4S4] provides electrons along with the hydrolysis of adenosine triphosphate (ATP).9,10 For an MoFe protein, numerous studies have confirmed that FeMo-co is the site where substrate reduction occurs,11–14 and it has been proposed that [Fe8S7] plays a pivotal role in transferring electrons between the iron protein and FeMo-co.15–17 Therefore, it is essential to demonstrate the redox states of M/P-clusters so as to understand their catalytic mechanism.

Nowadays, several oxidation states of P-clusters have been found, including the resting states PN, single-electron oxidized P1+ 18 and double-electron oxidized P2+ clusters.19,20 The most stable structures of P-clusters observed in protein crystals are PN and P2+. Their conformations can be transformed reversibly in the presence of reductant and oxidant.21 With its unstable thermodynamics, P1+ is a transient state whose crystal data was reported as PDB entry 6CDK with 60% completion.22 However, it has the probability of being a mixture of P1+ and P2+ according to quantum refinement calculations.23 Further oxidation states of P3+ and the others have been observed, but only PN, P1+ and P2+ were reported to be relevant to the catalytic cycle process.20,24 In a previous report, the “Deficit-spending” model proposed that FeMo-co obtains one electron from PN at the moment an iron protein interacts with an MoFe protein. Meanwhile, PN is turned to P1+ which is then rapidly refilled back to PN by electronic delivery from [Fe4S4],7 supposing no involvement of P2+ in this mechanism. However, recent work has shown that a P-cluster performs as P2+ while N2 coordinates with FeMo-co, implying that P2+ may play an important role in delivering electrons.25 Theoretical calculations have also proposed the catalytic involvement of P2+ in the density of states.26 Obviously, the roles of all oxidation states of the P-cluster are still uncertain, and the oxidation states of its irons are important for understanding the potential electron transfer sites and pathway.

The oxidation states of P-clusters have been proposed from electron paramagnetic resonance (EPR) and Mössbauer spectra,18,20,27–29 which enumerated all kinds of signals and possible spin states of irons in PN, P1+ and P2+ clusters. These early studies indicate that the resting state PN is all-ferrous.29,30 P1+ is the one-electron oxidized state of PN, and correspondingly, P2+ is commonly considered to come from double-electron oxidation.18 Later, X-ray crystallography revealed three different conformations of P-clusters as PN, P2+ 8 and P1+.22 Magnetic circular dichroism (MCD) was also applied to suggest the capability of electron delivery by the P-cluster.31 Nowadays, theoretical calculations with density functional theory (DFT)23 have provided the viewpoint that Fe6 and Fe7 are the most oxidized irons, and many-electron quantum wavefunction simulations illustrate the possible spin states and electronic structure of the P-cluster in plenty of aspects based on crystal structures.26 However, the detailed valence assignment of the P-cluster has not been analyzed specifically and agreed thus far. It will be helpful to extrapolate which irons play major roles in transferring electrons as the deficit-spending model describes. This inspired us to analyze the oxidation states of each iron in the P-cluster from the point of view of protein structures by the bond valence method.

The bond valence method was first used to analyze inorganic crystal structures32–36 and was gradually applied in other fields.37–41 It can be traced back historically to a proposal by Pauling.42 It is a classic and valid approach for assessing the charge between a metal atom and its bound coordinated atoms, and has proved an effective method to evaluate the electron density in a delocalized system43 and the oxidation states of metals in a metalloprotein.44–46 Up to now, the crystallographic structures of MoFe proteins deposited in the PDB have supplied sufficient bond data for M- and P-clusters. We have used this method to evaluate the valences of molybdenum(iii) and vanadium(iii) in FeMo/V-cofactors and the corresponding oxidation states of seven irons.46 In this work, we try to use the bond valence method to analyze the oxidation states of irons in P-clusters and to explore the function of PN in electron delivery between [Fe4S4] and FeMo-cofactor from different oxidized iron sites.

2. Calculation method

Bond valence sums (BVSs) were calculated using eqn (2), as shown below:

2. 2
2. 3

Si represents the calculated bond valence sum of each iron, and St in eqn (3) refers to the calculated valence sum of all eight Fe1–Fe8 irons (abbreviated to 8Fe below) in the P-cluster. The term rij is the bond distance between metal i (Fe) and ligand j (S/O/N/C), and B is commonly related to the softness of the bond47 and used as a constant equal to 0.37 Å.32R0 is a constant for a specific bond and varies with the assumed metal valence (Fen+) and coordinated atom as shown in Table 1. The values of R0 can be viewed on the web.48–50

The values of R0 corresponding to different types of bonds in P-clusters and their simulations.

M–L bonds R 0 (Å) M–L bonds R 0 (Å)
Fe2+–S 2.120 51 Fe3+–S 2.149 32
Fe2+–O 1.715 52 Fe3+–O 1.749 52
Fe2+–N 1.769 53 Fe3+–N 1.815 53
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For P-clusters in Mo/VFe proteins, rij is measured from crystal structures of Mo/VFe proteins deposited in the RCSB Protein Data Bank (PDB), which presently contains data on 119 P-clusters in 53 MoFe proteins and 10 P-clusters in 5 VFe proteins. For 14 PN-type model compounds, rij is acquired from the Cambridge Crystallographic Data Centre (CCDC) and measured by Pymol. The valences of Fe atoms were calculated by using R0 (+n) that corresponds to Fe2+ and Fe3+ coordinated with different ligands. All rij values and their resulting Si values are estimated to the third decimal place. Detailed bond valence calculations of all PDB entries and model compounds are given in Tables S4–S116.†

As an evaluation index, the absolute deviation |d| (d = Si − n, n is expected valence +2 or +3 for Fe atoms) represents the discrepancy between the calculated and expected valences, showing the fitting effects of R0 (+2) and R0 (+3). Due to the electron delocalization in P-clusters,54 some valences calculated with R0 (+2) and R0 (+3) show similar values of |d|. In this situation, iron valences can properly be regarded as mixed valence rather than integral valence. When the differences in |d| between R0 (+2) and R0 (+3) are distinct, the oxidation states of iron atoms should be assigned as the valence which has the smaller and more suitable value of |d|. The calculated valence sums of Fe1–Fe8 (St) also contrast with the assumed 8Fe all-ferrous valences which sum to “16” and all-ferric valences which sum to “24”. The resulting values of |d| shown in Fig. 2a and 4a imply the possible numbers of Fe3+ covered in this electron delocalization system and the total electrons reserved in P-clusters.

Fig. 2. The values of |d| of (a) total eight irons and (b–i) Fe1 to Fe8 in PN of FeMo proteins in terms of R0 (+2) (black) and R0 (+3) (red) respectively. Resolution is on the horizontal axis and the value of |d| is on the vertical axis. Some unusual PN data (PDB entries: 1M1Y, 2AFI, 6BBL, 6OP1, 6OP2, 6OP4) are excluded.

Fig. 2

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Fig. 4. (a) |d| calculated for R0 (+2) (black) and R0 (+3) (red) of Fe1–Fe8 in PN from 5 VFe proteins; (b) the weighted average |d| calculated for R0 (+2) (black) and R0 (+3) (red) of Fe1–Fe8 in PN from MoFe proteins.

Fig. 4

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As shown in Fig. 1b, in P1+, after single-electron oxidation of PN, Fe6 moves away from the central hexa-coordinated S1 atom and coordinates with the O atom in nearby amino acids such as Serβ188 in the MoFe protein of Azotobacter vinelandii (Av). With further one-electron oxidation, as shown in Fig. 1c, Fe5 in P2+ leaves the central S1 and bonds with the backbone amide N atom of Cysα88 in Av. Thus, the bonds of Fe5–S1 and Fe6–S1 are disconnected and Fe5–N and Fe6–O are formed in P1+ and P2+ respectively. Due to there being only a small number of deposited PDB entries containing P2+ or superposition of PN/2+, where two oxidation states coexist in P-clusters, we focus on researching the abundant data on PN and pick out the part relating to PN in the superposition structure, such as 3U7Q which is in the PN/2+ mixed state. The only data assigned as P1+ in MoFe protein (PDB entry: 6CDK) was also abandoned, because of the transient existence of P1+, dubious Fe–Fe and Fe–S bond distances23 and incompleteness of the crystal data.22

Fig. 1. Molecular structures of PN (a), P1+ cluster (b) (PDB entry: 6CDK) and P2+ (c) (PDB entry: 3U7Q) in MoFe proteins, and model compound of PN cluster (d) (CSD refcode: MUFQUA). Colors are Fe in green, S in yellow, O in red, N in blue, Si in plum and C in black.

Fig. 1

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To achieve reasonable big data analysis of bond valences, on the one hand, we carefully picked out these valid data in terms of protein structures. The P-clusters (PDB entries: 1M34, 5CX1, 5VQ4) which were not clearly assigned as PN/1+ in their conformational structures, were recognized as PN according to the reductive environments of protein purification, as Table S2† illustrates. Those data from unreasonable models (PDB entries: 1MIO, 3K1A) or structures containing deficient atoms (PDB entry: 6O7S) were abandoned, as shown in Table S3.† The protein data with unusually short Fe–S bonds (PDB entries: 1M1Y, 2AFI, 6BBL, 6OP1, 6OP2, 6OP4) which result in faulty St of 8Fe by using R0 (+2) above or approximating to 24 were not included in the analysis.

On the other hand, the bond valence method is empirical and has a great demand for high-precision data for bond distances. Thus, to deduce a more reasonable bond valence for each iron Si from all calculated P-clusters, it is crucial to select a suitable weighting formulation that includes resolutions of PDB data as weighting factors. Considering that a smaller value of the resolution Ai should have a higher weight wi, each wi(Ai) of PDB entries should be set as a function of the reciprocal resolution. It is appropriate to modify the inverse distance weighted (IDW) interpolation method to set a series of weights:

2. 4
2. 5

Univariate IDW interpolation is used widely by earth scientists in geochemistry.55–59 The character of eqn (4) is the same as the basic principle of IDW:60 the calculated bond valences Si from smaller values of resolution Ai such as 3U7Q have a greater influence on the weighted average valence than those from larger values of resolution, such as 1M34. Inline graphic in eqn (5) is the weighted average of the calculated valences Si of different Fe atoms from all analyzed PN clusters. N is the number of samples, including 69 PN clusters of MoFe proteins and 10 PN clusters of VFe proteins. Inline graphic is actually equal to 1 in this equation. Parameter p is an exponential parameter usually set to around 0.5–3.0 by the user.55,61 By comparing different wi by using different p values from a small amount of VFe protein data as shown in Table 2, we adopt p = 1,62 which also generate balanced wi for MoFe protein data of all various resolutions as in Fig. S1.†

The values of wi obtained by adopting different p in P-clusters of VFe proteins along with different resolutions.

PDB entries Res (Å) w i
p = 0.5 p = 1 p = 2 p = 3
7ADR 1.00 0.106 0.112 0.123 0.135
7ADY 1.05 0.103 0.106 0.112 0.116
7AIZ 1.05 0.103 0.106 0.112 0.116
6FEA 1.20 0.097 0.093 0.086 0.078
5N6Y 1.35 0.091 0.083 0.068 0.055
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Detailed supplemental illustrations of IDW, PDB classifications and bond valence calculations of all the above adopted and abandoned P-clusters can be seen in the ESI.†

3. Results and discussion

3.1. Criteria for valence assignment from 14 PN model compounds

Since BVS is an empirical method and considering the electron delocalization existing in an Fe–S cluster system, it is necessary to set a value of D as a valence assignment criterion to identify different integral and mixed valences. We consider that the new criterion applied to P-clusters could refer to the BVS results of the PN model compounds by using the same R0 parameter. 14 model compounds of PN have been selected from the Cambridge Crystallographic Data Centre (CCDC) and calculated by BVS. As shown in Fig. 1d, these model compounds have the same coordinated sites as natural PN. From the viewpoint of electronic structures, the PN model compounds have the same electron delocalization as natural PN, and are calculated by BVS as 2Fe3+6Fe2+, which is consistent with previous reports.63–65 After optimal selections of two calculated valences by using R0 (+2) and R0 (+3), two Fe(iii) and irons with mixed valences commonly exist in model compounds, as shown in Table 3. In a previous article about model compounds, the terminal Fe1 and Fe5 of each doublet were proved to be Fe3+,64–67 which is completely consistent with the calculated values.

The optimal calculated bond valences of Fe atoms in 14 model compounds obtained by using R0 (+2) or R0 (+3). Numbers of irons assigned different valences by using different presumed D are shown below.

CSD Refcodes Fe(1) Fe(2) Fe(3) Fe(4) Fe(5) Fe(6) Fe(7) Fe(8) Sum n(+3) : n(+2) : n(|d| > 0.25) n(+3) : n(+2) : n(|d| > 0.3) n(+3) : n(+2) : n(|d| > 0.35)
DUGNEZ 2.809 2.217 2.213 1.944 2.826 2.196 2.243 1.925 18.373 2 : 6 : 0 2 : 6 : 0 2 : 6 : 0
MUFPOT 2.814 2.234 2.145 2.608 2.842 2.249 2.140 2.606 19.639 2 : 4 : 2 2 : 4 : 2 2 : 4 : 2
MUFPUZ 2.804 2.217 2.146 2.322 2.804 2.217 2.146 2.322 18.978 2 : 4 : 2 2 : 4 : 2 2 : 6 : 0
MUFQAG 2.813 2.144 2.239 2.392 2.794 2.160 2.216 2.599 19.357 2 : 4 : 2 2 : 4 : 2 2 : 4 : 2
MUFQEK 2.809 2.232 2.169 2.628 2.886 2.277 2.153 2.607 19.760 2 : 3 : 3 2 : 4 : 2 2 : 4 : 2
MUFQIO 2.841 2.241 2.162 2.362 2.841 2.241 2.162 2.362 19.211 2 : 4 : 2 2 : 4 : 2 2 : 4 : 2
MUFQOU 2.898 2.212 2.201 2.660 2.764 2.228 2.185 2.347 19.495 2 : 4 : 2 2 : 4 : 2 3 : 5 : 0
MUFQUA 3.028 2.196 2.190 2.640 2.960 2.280 2.236 2.624 20.153 2 : 3 : 3 2 : 4 : 2 2 : 4 : 2
MUFRAH 2.983 2.206 2.198 2.267 2.932 2.226 2.236 2.361 19.409 2 : 4 : 2 2 : 5 : 1 2 : 5 : 1
MUFREL 3.000 2.268 2.188 2.335 3.004 2.290 2.350 2.166 19.601 2 : 2 : 4 2 : 4 : 2 2 : 6 : 0
NIFWOQ 2.855 2.139 2.267 2.350 2.855 2.139 2.267 2.350 19.223 2 : 2 : 4 2 : 4 : 2 2 : 6 : 0
NIFWUW 2.805 2.192 2.118 2.393 2.752 2.288 2.220 2.634 19.402 2 : 3 : 3 2 : 4 : 2 2 : 4 : 2
NIFXAD 2.840 2.230 2.196 2.620 2.840 2.230 2.196 2.620 19.772 2 : 4 : 2 2 : 4 : 2 2 : 4 : 2
WUZDAW 2.829 2.239 2.156 2.677 2.829 2.239 2.156 2.677 19.803 2 : 4 : 2 2 : 4 : 2 4 : 4 : 0
Average 2.866 2.212 2.185 2.443 2.852 2.233 2.208 2.443 19.441 2 : 4 : 2 2 : 4 : 2 2 : 4 : 2
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Although Thorp had stated that BVS values calculated from Brown's distance were reliable to ±0.25 units,44,53 we think that the electron delocalization in P-clusters compared with inorganic crystals requires a larger error-tolerance interval. In Table 3, we presumed three acceptable D values of 0.25, 0.3 and 0.35 as valence assignment criteria to compare the different valence assignments of iron. When the absolute deviation |d| between the calculated valence and the assumed valence is less than the valence assignment criterion D, such as 0.25, the irons are assumed to have valence Fe2+/3+; otherwise they ought to be assigned as having uncertainly mixed valence like Fe2.5+.

In Table 3, Fe4 and Fe8 sometimes show the character of mixed valence, which may be due to their spatial locations being adjacent to ferric Fe1 and Fe5. When choosing D as 0.25, several CSD entries like MUFREL and NIFWOQ show that Fe2/3/6/7 are assigned as mixed valences, which are obviously big deviations from the actual conclusion of 2Fe3+6Fe2+. If we set our sights on D = 0.35, this high error tolerance leads to another completely unreasonable conclusion that three or four irons(III) exist in PN model compounds (CSD codes: MUFQOU and WUZDAW).

In contrast, adopting 0.3 as the valence assignment criterion D, we find that the calculated and observed values are in good agreement, except for one or two irons with mixed valence. As discussed above, it is more credible for P-clusters to take a valence assignment criterion D = 0.3, according to the deviation calibration with the 14 most structurally similar PN model compounds. Thus, the following discussions about valence distributions of PN in Mo/VFe nitrogenases are based on this adopted criterion D.

3.2. Valence analyses of PN clusters in MoFe proteins

Fig. 2 shows the absolute deviations |d| of all 8 irons and each iron between calculated and assumed valences of PN in the resting state at a resolution of 2.3 Å. Detailed calculated results of each iron are shown in Table 4. In Fig. 2a, the discrepancies in the total valences of 8Fe between groups of R0 (+2) (black) and R0 (+3) (red) are obvious within a resolution of 1.6 Å. The weighted average value of |d| in the group of R0 (+2) is 2.15 if we assume PN is all-ferrous, and the corresponding |d| in the group of R0 (+3) is 4.47 when PN is assumed to be all-ferric. The smaller deviation calculated from all-ferrous parameters indicates that PN has a strong reductive property as previously reported22 and could undertake the function of delivering electrons.Fig. 2(b–i)show the absolute deviations |d| between calculated and presumed valences of each Fe, by using the parameters of R0 (+2) and R0 (+3) at different resolutions.

The bond valence sums calculated with R0 for Fe2+ and Fe3+ in valid P-cluster data, respectively, including 69 PN of 30 MoFe proteins and 10 PN of 5 VFe proteins. PDB entries with PN/2+ superposition are focused on the part of PN.

PDB codes Res (Å) R 0 (+2) R 0 (+3)
Fe1 Fe2 Fe3 Fe4 Fe5 Fe6 Fe7 Fe8 S t(8Fe) Fe1 Fe2 Fe3 Fe4 Fe5 Fe6 Fe7 Fe8 S t(8Fe)
P N in MoFe proteins
3U7Q 4 1.00 2.259 2.200 2.527 2.365 2.097 1.897 2.320 2.356 18.022 2.444 2.379 2.734 2.557 2.268 2.052 2.510 2.548 19.492
(Modeled as PN) 2.282 2.199 2.532 2.362 2.139 1.866 2.305 2.352 18.037 2.468 2.378 2.738 2.555 2.313 2.018 2.493 2.544 19.508
4WES 72 1.08 2.218 2.135 2.426 2.273 2.050 1.814 2.367 2.345 17.627 2.399 2.309 2.623 2.458 2.217 1.962 2.560 2.536 19.064
(Modeled as PN) 2.207 2.135 2.413 2.276 2.079 1.884 2.372 2.373 17.741 2.387 2.309 2.610 2.462 2.249 2.038 2.565 2.567 19.187
1M1N 3 1.16 2.198 2.197 2.426 2.364 2.201 2.079 2.281 2.298 18.043 2.377 2.376 2.624 2.556 2.381 2.248 2.467 2.486 19.515
2.225 2.234 2.409 2.376 2.182 2.098 2.239 2.294 18.057 2.407 2.416 2.606 2.569 2.360 2.269 2.421 2.481 19.530
2.211 2.203 2.440 2.336 2.237 2.106 2.308 2.275 18.117 2.392 2.383 2.639 2.527 2.420 2.278 2.496 2.461 19.594
2.241 2.260 2.482 2.392 2.193 2.084 2.254 2.293 18.198 2.423 2.444 2.684 2.587 2.372 2.254 2.437 2.480 19.682
7JRF 73 1.33 2.199 2.220 2.447 2.334 2.112 2.094 2.215 2.258 17.879 2.378 2.401 2.647 2.524 2.285 2.265 2.395 2.442 19.338
2.175 2.196 2.439 2.355 2.151 2.100 2.201 2.271 17.888 2.352 2.375 2.638 2.547 2.326 2.272 2.380 2.456 19.347
4TKU 74 1.43 2.317 2.244 2.563 2.372 2.269 2.136 2.231 2.316 18.448 2.436 2.414 2.745 2.584 2.437 2.331 2.435 2.478 19.860
2.253 2.232 2.538 2.389 2.253 2.155 2.251 2.291 18.363 2.506 2.427 2.772 2.565 2.454 2.310 2.413 2.505 19.952
4TKV 74 1.50 2.347 2.314 2.571 2.420 2.326 2.187 2.315 2.389 18.869 2.538 2.503 2.781 2.617 2.516 2.365 2.504 2.584 20.408
2.323 2.361 2.623 2.371 2.314 2.092 2.251 2.269 18.603 2.513 2.554 2.837 2.564 2.502 2.262 2.434 2.454 20.120
5BVH 75 1.53 2.231 2.291 2.545 2.364 2.220 2.140 2.280 2.288 18.360 2.413 2.478 2.753 2.557 2.401 2.315 2.466 2.475 19.857
2.246 2.323 2.538 2.369 2.229 2.155 2.275 2.307 18.442 2.429 2.512 2.745 2.562 2.410 2.331 2.461 2.495 19.946
1QGU 76 1.60 2.193 2.144 2.206 2.308 2.123 2.059 2.110 2.202 17.344 2.372 2.319 2.386 2.497 2.296 2.227 2.282 2.381 18.759
2.061 2.095 2.227 2.193 2.112 2.085 2.187 2.288 17.247 2.229 2.265 2.408 2.372 2.284 2.255 2.365 2.475 18.653
1QH8 76 1.60 2.122 2.112 2.316 2.253 2.108 1.987 2.369 2.465 17.731 2.295 2.285 2.504 2.437 2.280 2.148 2.563 2.666 19.177
(Modeled as PN) 2.187 2.196 2.345 2.317 2.123 1.948 2.281 2.413 17.810 2.366 2.375 2.536 2.506 2.297 2.107 2.467 2.610 19.262
5BVG 75 1.60 2.192 2.349 2.579 2.380 2.296 2.169 2.344 2.337 18.647 2.192 2.349 2.579 2.380 2.296 2.169 2.344 2.337 18.647
2.158 2.266 2.565 2.373 2.295 2.095 2.315 2.263 18.329 2.158 2.266 2.565 2.373 2.295 2.095 2.315 2.263 18.329
6OP3 77 1.60 2.630 2.557 2.729 2.649 2.206 2.077 2.117 2.275 19.241 2.844 2.766 2.952 2.865 2.386 2.247 2.289 2.461 20.810
2.503 2.449 2.715 2.738 2.225 2.098 2.197 2.284 19.209 2.707 2.648 2.936 2.962 2.407 2.269 2.376 2.470 20.775
graphic file with name d1ra08507g-t7.jpg 2.247 2.239 2.482 2.368 2.183 2.048 2.271 2.314 18.152 2.418 2.409 2.669 2.547 2.348 2.203 2.443 2.490 19.526
Weighted |d| 0.247 0.239 0.482 0.368 0.183 0.048 0.271 0.314 2.152 0.582 0.591 0.331 0.453 0.652 0.797 0.557 0.510 4.474
6O7P 78 1.70 2.356 2.354 2.590 2.303 2.603 2.124 2.236 2.272 18.839 2.475 2.543 2.901 2.507 2.691 2.401 2.474 2.487 20.477
2.288 2.351 2.683 2.318 2.488 2.220 2.287 2.299 18.934 2.548 2.546 2.801 2.491 2.815 2.297 2.419 2.457 20.375
5VQ3 79 1.72 2.246 2.179 2.430 2.420 2.163 1.986 2.389 2.228 18.041 2.429 2.356 2.628 2.617 2.339 2.148 2.583 2.410 19.511
2.174 2.199 2.458 2.299 2.145 2.109 2.374 2.378 18.135 2.351 2.379 2.658 2.486 2.320 2.281 2.567 2.572 19.614
6VXT 25 1.74 2.201 2.056 2.244 2.355 2.291 1.968 1.938 2.042 17.095 2.381 2.224 2.427 2.547 2.478 2.128 2.096 2.209 18.490
2.338 2.083 2.101 2.307 2.327 1.959 2.071 2.010 17.196 2.529 2.253 2.272 2.495 2.517 2.119 2.240 2.174 18.599
5CX1 80 1.75 2.348 2.333 2.526 2.222 2.293 2.089 2.329 2.296 18.436 2.540 2.523 2.732 2.403 2.480 2.259 2.519 2.484 19.940
2.393 2.316 2.468 2.292 2.209 2.170 2.370 2.315 18.535 2.588 2.505 2.669 2.479 2.389 2.347 2.564 2.504 20.046
2.249 2.330 2.450 2.206 2.270 2.168 2.319 2.322 18.315 2.433 2.520 2.650 2.386 2.455 2.345 2.508 2.511 19.808
2.193 2.389 2.555 2.381 2.042 2.097 2.290 2.217 18.165 2.371 2.584 2.764 2.575 2.209 2.268 2.477 2.398 19.647
2.270 2.315 2.581 2.222 2.302 2.135 2.389 2.284 18.498 2.455 2.504 2.792 2.403 2.489 2.309 2.584 2.470 20.006
2.426 2.433 2.567 2.405 2.186 2.047 2.341 2.336 18.741 2.624 2.632 2.776 2.601 2.364 2.214 2.532 2.526 20.269
2.256 2.407 2.476 2.370 2.308 2.153 2.309 2.285 18.563 2.440 2.603 2.678 2.563 2.496 2.328 2.497 2.471 20.076
2.344 2.377 2.538 2.299 2.126 2.164 2.312 2.306 18.466 2.535 2.571 2.745 2.486 2.300 2.340 2.500 2.494 19.972
5KOH 81 1.83 2.248 2.314 2.572 2.414 2.213 2.126 2.336 2.305 18.528 2.431 2.502 2.782 2.611 2.393 2.300 2.527 2.493 20.038
2.214 2.330 2.516 2.420 2.175 2.082 2.375 2.291 18.405 2.395 2.520 2.722 2.617 2.353 2.252 2.569 2.478 19.905
5VPW 79 1.85 2.233 2.197 2.423 2.374 2.122 1.919 2.239 2.234 17.740 2.493 2.543 2.665 2.606 2.230 2.037 2.539 2.395 19.508
2.305 2.351 2.464 2.409 2.062 1.884 2.347 2.215 18.037 2.415 2.376 2.620 2.567 2.295 2.075 2.421 2.416 19.186
6O7O 78 1.89 2.352 2.239 2.356 2.416 2.374 2.200 2.456 2.215 18.608 2.504 2.568 2.822 2.633 2.591 2.332 2.685 2.463 20.599
2.316 2.374 2.610 2.434 2.396 2.157 2.483 2.277 19.046 2.544 2.422 2.548 2.613 2.567 2.379 2.657 2.396 20.125
1H1L 82 1.90 2.077 2.149 2.317 2.341 1.991 2.039 2.213 2.230 17.358 2.246 2.324 2.506 2.532 2.154 2.206 2.393 2.412 18.773
2.101 2.225 2.514 2.438 2.120 1.964 2.141 2.235 17.738 2.272 2.406 2.719 2.637 2.293 2.124 2.315 2.417 19.184
4WZA 83 1.90 2.085 2.068 2.253 2.299 2.214 2.001 2.047 2.204 17.171 2.255 2.236 2.437 2.486 2.395 2.164 2.214 2.383 18.571
2.119 2.090 2.308 2.271 2.126 1.988 2.018 2.174 17.094 2.292 2.261 2.497 2.456 2.300 2.150 2.182 2.351 18.488
4WZB 84 1.90 2.137 2.008 2.213 2.266 2.029 1.903 1.966 2.139 16.660 2.311 2.171 2.394 2.451 2.195 2.058 2.126 2.314 18.019
2.165 1.956 2.278 2.197 2.211 1.844 2.083 2.074 16.809 2.341 2.115 2.464 2.376 2.391 1.994 2.253 2.243 18.179
4WNA 85 2.00 2.198 2.244 2.587 2.590 2.158 1.914 2.361 2.478 18.530 2.377 2.426 2.798 2.801 2.334 2.070 2.553 2.680 20.040
(Modeled as PN) 2.123 2.408 2.742 2.589 2.217 1.967 2.413 2.399 18.857 2.296 2.604 2.965 2.800 2.398 2.127 2.610 2.594 20.394
6O7Q 78 2.00 2.117 2.227 2.530 2.126 2.282 1.987 2.251 2.357 17.876 2.241 2.454 2.784 2.435 2.527 1.988 2.461 2.471 19.361
2.072 2.269 2.574 2.252 2.337 1.838 2.276 2.285 17.902 2.290 2.409 2.736 2.299 2.468 2.149 2.434 2.549 19.334
3MIN 8 2.03 3.031 2.536 2.829 3.226 2.763 2.223 2.590 2.306 21.505 3.279 2.743 3.060 3.489 2.988 2.405 2.801 2.494 23.259
2.671 2.324 2.803 2.593 2.400 2.021 2.494 2.536 19.841 2.889 2.514 3.031 2.804 2.595 2.186 2.697 2.742 21.459
2AFH 84 2.10 2.595 2.045 2.639 3.012 3.043 2.168 2.427 2.894 20.823 2.807 2.211 2.854 3.257 3.292 2.345 2.624 3.130 22.520
3.039 2.061 2.720 3.067 2.938 2.064 2.437 3.064 21.388 3.287 2.229 2.942 3.317 3.178 2.232 2.635 3.313 23.132
6O7L 78 2.26 2.266 2.391 2.775 2.528 2.976 2.179 1.679 2.623 19.417 2.450 2.586 3.002 2.734 3.219 2.357 1.815 2.836 21.000
2.153 2.151 2.815 2.394 2.331 2.232 1.853 2.238 18.167 2.329 2.326 3.044 2.589 2.521 2.414 2.005 2.420 19.648
6O7R 78 2.27 2.351 2.463 2.783 2.519 2.500 2.134 2.325 2.651 19.726 2.499 2.614 2.972 2.662 2.623 2.308 2.536 2.857 21.069
2.310 2.417 2.748 2.461 2.425 2.134 2.345 2.641 19.481 2.543 2.664 3.010 2.724 2.704 2.308 2.514 2.867 21.334
1L5H 86 2.30 2.815 2.552 2.367 2.297 2.258 1.783 2.541 2.402 19.015 3.045 2.760 2.560 2.485 2.442 1.928 2.748 2.598 20.565
1M34 87 2.30 2.326 2.492 2.480 2.466 2.321 1.990 2.376 2.480 18.931 2.516 2.695 2.683 2.667 2.511 2.152 2.570 2.682 20.474
2.507 2.531 2.592 2.914 2.325 1.840 2.438 2.323 19.468 2.712 2.737 2.803 3.151 2.514 1.990 2.637 2.512 21.055
2.556 2.564 2.577 2.656 2.154 2.130 2.624 2.166 19.425 2.764 2.773 2.787 2.872 2.329 2.304 2.838 2.342 21.008
2.589 2.645 2.597 2.590 2.161 1.995 2.428 2.317 19.323 2.800 2.861 2.809 2.801 2.337 2.158 2.626 2.506 20.898
5VQ4 79 2.30 2.218 2.287 2.646 2.493 2.040 1.737 2.457 2.635 18.513 2.399 2.474 2.862 2.697 2.206 1.878 2.657 2.850 20.023
2.026 2.236 2.548 2.483 2.039 1.736 2.130 2.286 17.484 2.191 2.419 2.755 2.686 2.206 1.877 2.304 2.472 18.910
graphic file with name d1ra08507g-t8.jpg 2.285 2.268 2.505 2.404 2.246 2.043 2.281 2.323 18.353 2.465 2.447 2.702 2.594 2.423 2.204 2.461 2.506 19.804
Weighted |d| 0.285 0.268 0.505 0.404 0.246 0.043 0.281 0.323 2.353 0.535 0.553 0.298 0.406 0.577 0.796 0.539 0.494 4.196
P N in VFe proteins
7ADR 88 1.00 2.154 2.085 2.324 2.290 2.183 2.050 2.397 2.365 17.846 2.329 2.255 2.513 2.476 2.361 2.217 2.592 2.557 19.301
(Modeled as PN) 2.137 2.081 2.315 2.265 2.204 2.084 2.405 2.372 17.862 2.312 2.250 2.504 2.450 2.384 2.254 2.601 2.565 19.319
7ADY 88 1.05 2.133 2.075 2.319 2.255 2.174 2.084 2.413 2.342 17.795 2.307 2.244 2.508 2.439 2.351 2.254 2.610 2.533 19.245
(Modeled as PN) 2.157 2.076 2.327 2.286 2.171 2.022 2.386 2.356 17.783 2.333 2.246 2.517 2.473 2.349 2.187 2.581 2.548 19.233
7AIZ 89 1.05 2.145 2.081 2.311 2.269 2.202 2.080 2.422 2.351 17.861 2.320 2.251 2.499 2.454 2.382 2.250 2.620 2.543 19.319
(Modeled as PN) 2.175 2.069 2.301 2.295 2.143 1.990 2.395 2.325 17.693 2.352 2.238 2.489 2.482 2.318 2.153 2.590 2.514 19.137
6FEA 90 1.20 2.155 2.103 2.344 2.320 2.213 2.046 2.438 2.411 18.030 2.331 2.274 2.535 2.509 2.393 2.213 2.637 2.608 19.500
(Modeled as PN) 2.181 2.108 2.383 2.295 2.253 2.072 2.457 2.400 18.148 2.359 2.279 2.578 2.482 2.437 2.240 2.657 2.595 19.627
5N6Y 91 1.35 2.249 2.145 2.432 2.324 2.300 2.176 2.511 2.461 18.599 2.433 2.320 2.631 2.513 2.488 2.353 2.716 2.662 20.115
2.192 2.165 2.336 2.269 2.261 2.115 2.449 2.430 18.216 2.371 2.341 2.526 2.453 2.445 2.288 2.648 2.628 19.701
graphic file with name d1ra08507g-t9.jpg 2.165 2.096 2.336 2.286 2.206 2.069 2.424 2.377 17.959 2.342 2.267 2.527 2.472 2.386 2.237 2.622 2.571 19.424
Weighted |d| 0.165 0.096 0.336 0.286 0.206 0.069 0.424 0.377 1.959 0.658 0.733 0.473 0.528 0.614 0.763 0.378 0.429 4.576
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For Fe1, it is clear that most deviations |d| of the two groups are separated by a value of 0.4 in Fig. 2b, with a weighted average value of 0.29 for group R0 (+2) and 0.54 for R0 (+3) within 2.3 Å resolution. As can be seen from Table 4, the difference |d| between the BVS of R0 (+2) and +2 valence even drops to 0.25 within a resolution of 1.6 Å, with both |d| of R0 (+2) below the adopted D, which implies that Fe1 tends to be ferrous and the assignment of Fe(ii) might be appropriate. For Fe2, the weighted average value of |d| is 0.27 for R0 (+2) and 0.55 for R0 (+3). Besides, Fe2 is prone to be iron(ii) whose |d| is 0.24, which is obviously below D = 0.30 in group R0 (+2) within the resolution of 1.6 Å. For Fe3, it is the iron which is most prone to be Fe(iii) compared with other irons, where its weighted average values of |d| for R0 (+2) and R0 (+3) are 0.51 and 0.30, respectively. However, compared with Fe3 which possesses a smaller deviation |d| of R0 (+3) than R0 (+2) in the overall data, the |d| of the two groups R0 (+2) and R0 (+3) for Fe4 partly overlap and are tangled up over the whole range of resolutions, where its weighted averages |d| are 0.40 and 0.41 for R0 (+2) and R0 (+3). This implies that Fe4 has a strong mixed valence character29,68 with a more oxidized state than Fe(ii), and the valence assignment of Fe4 could not be defined.

In the same way, Fe5, Fe6 and Fe7 are more inclined to be Fe2+ rather than Fe3+ in Fig. 2. The corresponding weighted average values of |d| of R0 (+2) and R0 (+3) are 0.25 and 0.58 for Fe5, 0.04 and 0.80 for Fe6, and 0.28 and 0.54 for Fe7, respectively. The deviations 0.18 and 0.05 for group R0 (+2) of Fe5 and Fe6 are even smaller than 0.2 within a resolution of 1.6 Å. However, unlike Fe5 or Fe6, as shown in Fig. 2f–g, the groups of R0 (+2) and R0 (+3) for Fe7 are separated by D but are not so distinct, which implies that its electrons are not well localized. The |d| values of Fe8 for R0 (+2) groups are 0.31 and 0.32, respectively, no matter whether the data are for resolutions below 1.6 Å or 2.3 Å. Referring to the aforementioned valence assignment criterion |D| of 0.3, Fe8 and Fe4 are alike and should be regarded as mixed valence, but Fe4 has a tendency to be more oxidative than Fe8. From Table 4, the weighted average valence sum of 8Fe is 18.353 by using R0 (+2), and most valence sums of 8Fe are distributed in the range of 17 to 19, resembling PN model compound DUGNEZ.66 The above discussions and the calculated results elucidate that resting state PN clusters in protein crystals contain one ferric and two iron atoms of mixed valence, and ought to have an oxidation state approximately equal to 6Fe(ii)2Fe(iii) with delocalized electrons. However, due to widespread electron delocalization, mixed valences existing in Fe3/4/8 influence their accurate valence assignments. Given the limitation of BVS applied in a mixed-valence system, we consider that Fe1/2/5/6/7 should be assigned to Fe2+ and Fe4/8 are prone to being mixed valence as Fe2.33+ or Fe2.5+, which has been reported in [MFe3S4] model compounds.70,71 Fe3 has a larger possibility of possessing states of Fe(iii) or Fe2.5+, and more oxidative mixed valences compared with Fe4/8.

According to Fig. 2, we can see that Fe3 and Fe6 are obviously the most oxidized and reduced iron atoms in PN. When changing our focus to the spatial locations of three clusters, as shown in Fig. 3, we found that Fe3 has the shortest 9.0 Å distance in the eight iron atoms to homocitrate in the M-cluster, which participates in catalysis as an electron-demander and takes charge of the reduction of the substrate. Fe4, which ranks as the second highest mixed valence in the eight irons, is also the iron second closest to the M-cluster. Besides, Fe6 with a distance of 15.0 Å is the nearest iron to the electron-donor [Fe4S4] which is responsible for the “backfill” electron transfer to P1+ in the deficit-spending mechanism. Similarly, Fe1/2, which side by side with Fe6 perform with an obviously reductive character, are the irons spatially second closest to the F-cluster.

Fig. 3. The spatial position between the F-cluster, PN cluster and FeMo-cofactor from PDB entry 4WZB in 1.9 Å,69 which simultaneously contains an MoFe protein and an Fe protein with the highest resolution in all deposited PDB data. Nearby amino acid residues are simplified.

Fig. 3

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From the perspective of PN structure, terminal Fe3 and Fe7 seem to be oxidized more easily, which is similar to the report that oxidation occurs preferentially at the peripheral iron sites Fe(1) and Fe(5) in PN model compounds.64 Nearby Fe3 and Fe7, Fe4 and Fe8 are also calculated out mixed valence as Fe(4) and Fe(8). By maintaining PN in reductive solution with excess reducing agents, plenty of experiments give the conclusion that PN clusters are all-ferrous according to Mössbauer and EPR at an early stage.20,28,29 But irons in model compounds of PN could have the character of 6Fe(ii)2Fe(iii), as reported. As Fig. 1d shows, structures of PN model compounds display the same coordinated sites as those amino acid residues bonding with natural PN. Interestingly, the terminal Fe(iii) sites Fe1 and Fe5 in PN model compounds correspond to terminal irons Fe3 and Fe7 in natural PN.64,65 Thus, partially oxidized PN model compounds could still maintain their original structure, which indicates that PN in the form of protein crystals has the possibility of being in the same oxidative state of 6Fe(ii)2Fe(iii) without structural change. On the other hand, the calculated results of the most accurate PDB entry 3U7Q indeed display consistent conclusions with the above discussion. Its Fe1, Fe2, Fe5 and Fe6 obviously approach Fe2+ and Fe3 tends to Fe3+ with an average value of 2.736 in the group of R0 (+3), where different degrees of mixed valence are distributed in electron-delocalized Fe4, Fe7 and Fe8.

Thus, we could conclude that the oxidation states of the eight irons in PN of MoFe protein crystals are different and not completely all-ferrous. In protein crystals, Fe1, Fe2, Fe5 and Fe6 still perform with strong reductive character as in a reductive bio-environment, but Fe3 and Fe4/8/7 are more oxidized than other irons in consequence, even though PN is reduced in the initial stage.

3.3. Valence analyses of PN clusters in VFe proteins

From Fig. 4, P-clusters of five VFe proteins have similar degrees of oxidation states in each iron. The average valence sums St 18.407 of 5N6Y and 18.089 of 6FEA by R0 (+2) indicate that P-clusters of the latter are more reduced than those of the former as a whole, which is consistent with the discovery we also made before for FeMo-cofactors.46

For Fe1, Fe2, Fe4, Fe5 and Fe6 in Table 4 and Fig. 4a, their sideways |d| of groups R0 (+2) are below 0.3 and smaller than those of R0 (+3), indicating that the assignment of iron(ii) is appropriate, while Fe4 shows a small degree of mixed valence. Compared with the PN of MoFe proteins in Fig. 4b, P-clusters in VFe proteins have the similarity that Fe3, Fe7 and Fe8 perform with obvious characters of mixed valences while there are dramatic differences in Fe7 and Fe8 which are more oxidized than Fe3. In Table 4 it can be seen that Fe7 and Fe8 with strong electron delocalization have similar |d| of 0.38 and 0.43 by using R0 (+3), and 0.42 and 0.38 by using R0 (+2), which manifest the tendency of Fe7 and Fe8 for being high mixed valence. Opposite to MoFe proteins, Fe3 in VFe proteins are more inclined to be iron(ii) in mixed valence, with |d| of 0.34 and 0.47 by using R0 (+2) and R0 (+3), respectively. From the perspective of the weighted average valence sum St (17.96) of 8Fe by R0 (+2), the PN in the VFe protein with a similar formal oxidation state of 6Fe(ii)2Fe(iii), is more reductive than the MoFe protein to a small degree. Although the core structure [Fe8S7] of the P-cluster is the same in MoFe and VFe proteins, it can be seen that their electron distributions of the P-cluster are apparently different, as the electron distribution of FeMo-co is also different from that of VFe-co.46 The above difference could be attributed to different structures between MoFe and VFe proteins, which may result in different electron transfer channels to induce the formation of the relevant iron oxidation states.

4. Conclusions

We have studied all deposited PN in 119 P-clusters of 53 MoFe PDB entries and 10 P-clusters of 5 VFe PDB entries with the bond valence method. In Mo/VFe protein crystals, PN clusters are all supposed formally to be 6Fe(ii)2Fe(iii). All of their Fe1, Fe2, Fe5 and Fe6 ought to be assigned as Fe2+, while the mixed valences Fe2.33+/2.5+ in Fe3, Fe4, Fe7 and Fe8 are differently distributed, probably due to their different protein structures. These reflect which Fe atoms have a tendency to maintain oxidized or reduced states of Mo/VFe proteins in crystal form. The calculated results of PN in crystals seem not to be the same as in traditional ideas that PN clusters are “all-ferrous” from those analyses in reductive solutions with excess reducing agents. In view of the spatial position of the MoFe protein crystal, the most oxidized Fe3 and reduced Fe6 are simultaneously the nearest irons to FeMo-co and [Fe4S4], respectively. It seems that Fe3 and Fe6 could function as the most convenient electron transfer sites. This potential difference in PN clusters might be more suitable to correlate with the other two F- and M-clusters as an important electron transfer station.

This work first applied the bond valence method in P-clusters of Mo/VFe proteins statistically, which provided a new perspective in the general electron distributions of P-clusters in nitrogenase, and might be widely applied to other metalloenzyme systems with electron delocalization. Our work delivers a much more detailed evaluation of the oxidation states of the eight irons in the P-cluster, adding a special story to research into nitrogenase. More insightful pursuits still need further investigations. All of these studies were built on predecessors' work that supplied sufficient crystal data of Mo/VFe proteins in the PDB to help educe reasonable results.

Conflicts of interest

The authors declare no competing financial interests.

Supplementary Material

RA-012-D1RA08507G-s001

Acknowledgments

We thank the National Natural Science Foundation of China (22179110) for generous financial support.

†

Electronic supplementary information (ESI) available: Details of bond distances and valence calculations of all relevant PDB entries in Tables S1–S116. See DOI: 10.1039/d1ra08507g

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