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. Author manuscript; available in PMC: 2019 Oct 25.
Published in final edited form as: Inorg Chem. 2019 Feb 6;58(4):2523–2532. doi: 10.1021/acs.inorgchem.8b03108

Preliminary Assignment of Protonated and Deprotonated Homocitrates in Extracted FeMo-cofactors by Comparisons with Molybdenum(IV) Lactates and Oxidovanadium Glycolates

Wan-Ting Jin a, Hongxin Wang b,c, Si-Yuan Wang a, Christie H Dapper d, Xing Li a, William E Newton d, Zhao-Hui Zhou a,*, Stephen P Cramer b,c,*
PMCID: PMC6813765  NIHMSID: NIHMS1055471  PMID: 30726074

Abstract

A similar pair of protonated and deprotonated mononuclear oxidovanadium glycolates [VO(Hglyc)(phen)(H2O)]Cl·2H2O (1) and [VO(glyc)(bpy)(H2O)] (2) and a mixed-(de)protonated oxidovanadium triglycolate (NH4)2[VO(Hglyc)2(glyc)]·H2O (3) were isolated and examined. The ≡C–O(H) (≡C–OH or ≡C–O) groups coordinated to vanadium were spectroscopically and structurally identified. The glycolate in 1 features a bidentate chelation through protonated α-hydroxy and α-carboxy groups, whereas the glycolate in 2 coordinates through deprotonated α-alkoxy and α-carboxy groups. The glycolates in 3 coordinate to vanadium through α-alkoxy or α-hydroxy and α-carboxy groups, and thus has both protonated ≡C–OH and deprotonated ≡C–O bonds simultaneously. Structural investigations revealed that the longer protonated V–Oα-hydroxy bonds [2.234(2) Å and 2.244(2) Å] in 1 and 3 are close to those of FeV-co 2.17 Å1 (FeMo-co 2.17 Å2), while deprotonated V–Oα-alkoxy bonds [2, 1.930(2); 3, 1.927(2) Å] were obviously shorter. This shows a similar elongated trend as the Mo–O distances in the previously reported deprotonated vs. protonated molybdenum lactates,3 and these vanadium and molybdenum complexes have the same local V/Mo-homocitrate structures as those of FeV/Mo-cos of nitrogenases. The IR spectra of these oxidovanadium and the previously synthesized molybdenum complexes including different substituted ≡C–O(H) model compounds show red-shifts for ≡C–OH vs. ≡C–O alternation, which further assign the two IR bands of extracted FeMo-co at 1084 and 1031 cm−1 to ≡C–O and ≡C–OH vibrations respectively. Although the structural data or IR spectra for some of the previously synthesized Mo/V complexes and extracted FeMo-co were measured earlier, this is the first time that the ≡C–O(H) coordinated peaks are assigned. The overall structural and IR results well suggest the co-existence of homocitrates coordinated with α-alkoxy (deprotonated) and α-hydroxy (protonated) groups in the extracted FeMo-co.

Keywords: Infrared spectroscopy, hydroxycarboxylate, FeMo-cofactor, FeV-cofactor, nitrogenase

Graphical Abstract

graphic file with name nihms-1055471-f0001.jpg

A similar pair of protonated and deprotonated mononuclear oxidovanadium glycolates [VO(Hglyc)(phen)(H2O)]Cl·2H2O (1) and [VO(glyc)(bpy)(H2O)] (2) and a mixed-(de)protonated oxidovanadium triglycolate (NH4)2[VO(Hglyc)2(glyc)]·H2O (3) were isolated. The structural comparison and IR spectra of these oxidovanadium complexes as well as the previously synthesized molybdenum complexes are cited for the IR assignment of ≡C–O(H) vibrations in extracted FeMo-cofactor, suggesting co-existence of homocitrates coordinated with α-alkoxy (deprotonated) and α-hydroxy (protonated) groups in the extracted FeMo-co.

INTRODUCTION

Nitrogenases catalyze the reduction of dinitrogen (N2) to ammonia (NH3) in nature. The enzymes have been extensively investigated and the structures of their catalytic active sites FeMo/V-cofactors (FeMo/V-cos) have been finally clarified as MoFe7S9C(cys)(Hhis)(R-homocit)2,4–6 and VFe7S8C(cys)(Hhis) (XO3)(R-homocit) (H4homocit = homocitric acid, X = C or N respectively, Hcys = cysteine, C3H7NO2S, Hhis = histidine, C6H9N3O2),1,7 where homocitrates coordinate with metal Mo or V via the oxygen atoms of α-alkoxy and α-carboxy groups and have a charge of −4.8 Spectroscopic studies with infrared spectroscopy (IR),9,10 magnetic-circular-dichroism spectroscopy (MCD),11,12 19F nuclear magnetic resonance spectroscopy (19F NMR),13 X-ray absorption spectroscopy (XAS),14–19 Mössbauer spectroscopy,20,21 electron-nuclear double resonance (ENDOR),22,23 electron spin echo envelope modulation (ESEEM),22,23 impulsive coherent vibrational spectroscopy (ICVS),24 nuclear resonance vibrational spectroscopy (NRVS),25,26 and electron paramagnetic resonance (EPR)20,27 show a low valence and paramagnetic nature for FeMo/V-cos. The charge on FeMo-co has been controversial as the metal oxidation states of FeMo-co were suggested as Mo(IV)6Fe(II)1Fe(III),28 Mo(IV)4Fe(II)3Fe(III),29 Mo(IV)2Fe(II)5Fe(III)30 and Mo(III)3Fe(II)4Fe(III) respectively.31,32 In addition to the FeMo/V-cos’ central structures, the local structure about the Mo/V-homocitrato coordination is critical for the nitrogenase studies. We have suggested a protonated model for FeMo/V-cos based on a computational study33 and protonated α-hydroxycarboxylato oxidovanadium complexes.34 Structural comparisons of glycolato and lactato molybdenum(IV) complexes and nitrogenases provide indirect evidence for the protonation of homocitrate in FeMo-co.3 In addition, there are several recent theoretical computational calculations for the favorite protonation states in α-alkoxy group of homocitrate ligand of FeMo-co.30,35–37

Most of the previous glycolate, lactate, malate, citrate or homocitrate bind to vanadium or molybdenum via α-alkoxy and α-carboxy, and/or β-carboxy groups,38–63 while only a small number of complexes were isolated coordinating via α-hydroxy (protonated) and α-carboxy groups.34,64–68 As the homocitrates in the cofactors have a bidentate coordination, protonated and deprotonated molybdenum/vanadium α-hydroxycarboxylates with the similar local structures are critical for comparisons with the FeMo/V-cos and for a better clarification of their coordination environments. In this publication, we report the successfully isolated protonated and deprotonated pair of mononuclear glycolato oxidovanadium complexes: [VO(Hglyc)(phen)(H2O)]Cl·2H2O (1), [VO(glyc)(bpy)(H2O)] (2) (H2glyc = glycolic acid, bpy = 2,2’-bipyridine, phen = 1,10-phenanthroline). Oxidovanadium triglycolate (NH4)2[VO(Hglyc)2(glyc)]·H2O (3) with a mixed-(de)protonated state was also synthesized and analyzed. All these complexes have a similar bidentate chelated local structure as the homocitrate has in FeV-co or FeMo-co. The infrared spectrum (IR) of the extracted FeMo-co9,69,70 has been re-examined. The structures and IR spectra of the above vanadium complexes (1 ~ 3) are compared with those of FeMo/V-cos, as well as compared with the protonated and deprotonated molybdenum(IV) lactates [MoIV3S4(PPh3)3(Hlact)2(lact)],62 Na2[MoIV3SO3(R,S-lact)3(im)3]·10H2O3,67 and different substituted ≡C–O(H) model compounds. Although the structural data or IR spectra for some of the previously synthesized Mo/V complexes were measured earlier, this is the first time that the ≡C–O(H) coordinated peaks are assigned. Via these comparisons, the protonated/deprotonated states of homocitrate in extracted FeMo-co are assigned experimentally and discussed systematically for the first time.

RESULTS AND DISCUSSION

Syntheses.

In the preparations of 1 and 2, the precursor V2O5 was reduced to VO2+ species by water in acidic solution under hydrothermal condition. The reactions of V2O5 with excess glycolic acid and N-chelated ligands are sensitive to the pH values. The deprotonated 2 was isolated at pH 3.0 in the presence of bipyridine, while the protonated 1 was obtained under low pH value of 1.0 in the presence of phenanthroline. The reactant of bipyridine or phenanthroline was not in excess. 3 was obtained from the reaction of VOSO4 with three equivalents of glycolic acid without the participation of N-chelated ligand.24,25 The synthesis of 3 was at room temperature but the crystallization was found sensitive to the temperature and the concentration of the reactants. Moreover, the effects of pH variations between 4 ~ 6 and the ratio of V:ligand (1:2 or 1:3) seem less crucial for the formation of 3 in comparison with the cases in 1 and 2. The complexes 1 and 3 are soluble in water, while 2 is insoluble.

Crystal Structures.

The ORTEP diagrams of 1 ~ 3 are shown in the Supplementary Information (SI†) - Figs. S1 ~ S3. The detailed X-ray crystallographic data and the selected bond distances and angles for 1 ~ 3 are listed as in Tables S1 ~ S3. X-ray crystallographic analyses reveal that the glycolate in 1 coordinates bidentately to vanadium atom via protonated α-hydroxy and α-carboxy groups, while the glycolate in neutral molecule 2 coordinates via deprotonated α-alkoxy and α-carboxy groups. The V(IV) ions in 1 and 2 exist in distorted octahedral geometries with N2O4 donor set. In 1, two nitrogen atoms of phenanthroline (N1 and N2) and one oxygen atom of water molecule (O1w), as well as the oxygen atom in α-carboxy group of glycolate (O2) occupy the four equatorial positions. The oxygen atom in α-hydroxy group of glycolate (O1) is at one axial position, trans to the terminal oxygen atom (O4) on the other axial site. The 2-D packing diagram of 1 is provided in Fig. S4, showing hydrophobic π–π interaction of phenanthroline. In 2, the oxygen atoms in α-alkoxy (O1) and α-carboxy groups (O2) are at two equatorial positions, while one coordinated water molecule (O1w) and one nitrogen atom of bpy (N1) occupy the other two equatorial positions. The other nitrogen atom of bpy (N2) is at one axial position and the terminal oxygen atom (O4) is at the final axial site. The 2-D packing diagram of 2 is given in Fig. S5, showing hydrophobic π–π interaction of bipyridine.

The vanadium ion in 3 coordinates with three glycolates, presenting a distorted octahedral geometry. One of the glycolates coordinates via α-alkoxy (O3) and α-carboxy (O4) groups, where the two oxygen atoms are at two equatorial positions. The second glycolate coordinates to vanadium via α-carboxy group (O5), leaving the α-hydroxy group (O10) free. The last glycolate coordinates via α-hydroxy (O1) and α-carboxy (O2) groups, where the α-hydroxy (O1) is trans to terminal oxygen atom (O6) on the final axial site. Coordinating with both of the protonated and deprotonated glycolates, 3 can serve as a mixed-(de)protonated model complex compared with 1 and 2. The anion structure of 3 is similar to the reported potassium salt K2[VO(Hglyc)2(glyc)]·H2O.64,65

The structural environments near the V-glycolate coordinations of 1 ~ 3 are shown in Fig. 1, and their hydrogen bonds are listed in Table S3. For 1, α-hydroxy group forms strong hydrogen bond with α-carboxy group [O1···O3a 2.71(1) Å, a (x + ½, -y + ½, z + ½)], and the coordinated water molecule forms strong hydrogen bonds with crystal water molecules [O1wa···O2wa 2.62(1) Å; O1wa···O3wa 2.58(1) Å]. The free chloride anions also connect with the lattice water molecules by hydrogen bonds [Cl1a···O3wa 3.03(1) Å; Cl1a···O2wb 3.05(1) Å, b (x + ½, -y + ½, z + 3/2); and Cl1a···O3wc 3.15(1) Å, c (x + ½, y - ½, z + 1)]. For 2, the coordinated water molecules form strong hydrogen bonds with α-alkoxy and α-carboxy groups [O1w···O1a 2.58(1) Å, a (-x + 1, -y, -z + 2); O1w···O3b 2.65(1) Å, b (-x + 1, y − ½, -z + 3/2)]. For 3, a mixed-(de)protonated state is formed in 3 as the strong interactions of hydrogen bonds are favourable for the proton exchange between α-alkoxy and α-hydroxy groups. The α-alkoxy O3 and α-hydroxy O1 form strong hydrogen bonds with α-hydroxy O1a and α-alkoxy O3a from the adjacent molecule respectively. The distance of hydrogen bond [O1···O3a 2.55(1) Å, a (1 − x, 1 − y, 1 − z)] is similar to the O···O distance (2.50 Å) calculated by a protonated QM/MM model.35

Figure 1.

Figure 1.

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The environment of [VO(Hglyc)(phen)(H2O)]Cl·2H2O (1) showing hydrogen bonds between α-hydroxy groups, water molecules and α-carboxy groups; [VO(glyc)(bpy)(H2O)] (2) showing hydrogen bonds between water molecules and α-alkoxy groups; (NH4)2[VO(Hglyc)2(glyc)]·H2O (3) showing hydrogen bonds between α-hydroxy groups and α-alkoxy groups. Ammonium ions and lattice water molecules were omitted for clarity.

The V−O distances of α-hydroxycarboxylato vanadium complexes vary systematically according to the bond types and the oxidation states of the vanadium ions. Theoretical bond valence calculations71,72 (Table S4) and EPR spectra (Figure S6) gave the valence of +4 for 1 ~ 3 respectively. As shown in Table 1, protonated and deprotonated oxidovanadium products 1 and 2 exhibit different V–O distances. The V1–O1α-hydroxy distance in 1 [2.231(2) Å] (protonated) is much longer than that of V1–O1α-alkoxy in 2 [1.931(2) Å] (deprotonated). In 3, the V1–O1α-hydroxy distance [2.244(2) Å] (protonated) is found longer than V1–O3α-alkoxy distance [1.927(2) Å] (deprotonated). The difference (~ 0.3 Å) between V–Oα-hydroxy and V–Oα-alkoxy distances can be attributed to the trans effect of V=O group and the equalization of electronic cloud density resulted from the protonation. The protonation contributes about 0.1 Å to the change of V–Oα-hydroxy distance when excluding the trans effect from V=O group, which is supported by the protonated complexes [VO(C5H9O3)2(C5H8N2)]67 [V1–O2 (protonated, axial site), 2.209(2) Å; V1–O5 (protonated, equatorial site), 2.023(2) Å] and [VO(H2cit)(phen)]2·6.5H2O [V1–O1 (protonated, axial site), 2.203(5) Å; V2–O11 (protonated, equatorial site), 2.026(5) Å].34 On the other hand, the trans effect contributing about 0.2 Å to the elongation of the V–Oα-hydroxy distance is also supported by the change of V–N distances, where the nitrogen atom on the trans position [2.316(2) Å] is longer than those of on the equatorial sites [2.129(2)av Å].

Table 1.

Comparisons of V/Mo–O bond distances (Å) in α-hydroxycarboxylato vanadium/molybdenum complexes.

Complexes M–O
(α-hydroxy)		 M–O
(α-alkoxy)		 M–O
(α-carboxy)
1 2.231(2) 1.997(2)
2 1.931(2) 2.018(2)
3 2.244(2) 1.927(2) 2.027(2)av
VIVO34,64–66 2.210(5)av 1.925(4)av 2.010(5)av
VIV2O238,39,43,44 2.020(5)av 2.019(6)av
VIV/V2O341 1.856(4)av 2.080(4)av
VV2O442,44–52,65 1.985(8)av 1.975(8)av
FeV-co1 (1.35 Å) 2.170av 2.112av
FeV-co7 (1.2 Å) 2.160av 2.104av
 
Mo0 (non-nature)68 2.273(8)av 2.233(8)av
MoIV 3,67 2.204(4)av 2.010(3)av 2.121(4)av
MoV 58,59,62,63 2.001(8)av 2.142(12)av
MoVI 53–61 1.959(8)av 2.203(7)av
FeMo-co2(1.0 Å) 2.171av 2.202av
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*

The subscript av represents an average value. The detail data can be available in Tables S5 ~ S7 in Supplementary Information (SI†).

Based on the MoIV/III and VIII proposed for FeMo-co and FeV-co,73–76 the V/Mo–O(H) distances of α-hydroxycarboxylato vanadium/molybdenum complexes with different coordination modes and different oxidation states are listed in Table 1 for comparisons with FeV/Mo-cos. The protonated VIV−Oα-hydroxy distances [2.210(5)av Å] are the closest distances to the corresponding V–Oα-hydroxy/α-alkoxy distance in homocitrato FeV-co (2.170av and 2.160av Å).1,7 While the other deprotonated V–Oα-alkoxy distances are shorter than that of FeV-co to different degrees, including the VIV–Oα-alkoxy distance [1.925(4)av Å], the pentavalent VV–Oα-alkoxy distance [1.856(4)av Å] in V2O3 cores, as well as the V–Oα-alkoxy distances (~ 2.0 Å) in dimeric V2O2 or V2O4 units. The VV–Oα-alkoxy distances are shorter than the VIV–Oα-alkoxy distances due to the higher oxidation state of the metal center. The V–Oα-alkoxy distances in V2O4 or V2O2 units are slightly longer than the aforementioned VIV–Oα-alkoxy and VV–Oα-alkoxy distances due to the bridging coordination modes, where the α-alkoxy group serves as bridging ligand. On the other hand, the distances between vanadium and α-carboxy groups (around 2.0 Å) are shorter than that of homocitrate in FeV-co (2.112av and 2.104av Å).1,7

From the Table 1, the protonated Mo–Oα-hydroxy distances of α-hydroxycarboxylato molybdenum complexes are about 0.2 Å longer than those of deprotonated Mo–Oα-alkoxy bonds as described previously.3 The MoIV–Oα-hydroxy distances of 2.179(4) Å and 2.228(4) Å in [MoIV3S4(PPh3)3(Hlact)2lact]67 are close to the value of 2.17av Å in FeMo-co,2 while the MoIV–Oα-alkoxy distances of 2.022(3)av Å in Na2[Mo3SO3(lact)3(im)3]·10H2O3 are obviously shorter. This is also true for the Mo0–Oα-hydroxy (non-nature) bonds, which are the longest protonated distances due to their lowest oxidation states. In comparison with the bond distances of these similar compounds, the vanadium/molybdenum atoms in cofactors might have weak interactions with homocitrates and protonated α-hydroxy coordinations (Figure 2).

Figure 2.

Figure 2.

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The structures of model vanadium/molybdenum hydroxycarboxylates with low oxidation states.

13C NMR Measurement.

Solution 13C NMR spectrum shown in Fig. 3 provides valuable information on the coordination environment and chemical behavior of 1. The resonances for α-carboxy (α-CO2) and α-hydroxy (α-COH) groups in 1 are at around 178.9 and 61.9 ppm respectively, showing downfield shifts compared with free glycolic acid (α-CO2 177.0 ppm; α-≡COH 60.2 ppm). The small shifts indicated that the protonated coordination environment of glycolate ligand in 1 was close to the free state, which implies a weak coordination between oxidovanadium ion and α-hydroxy group.

Figure 3.

Figure 3.

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Solution 13C NMR spectrum of [VO(Hglyc)(phen)(H2O)]Cl·2H2O (1) in D2O.

FT-IR Measurements.

The FT-infrared spectra of the solids 1 and 2 in the regions of 1800–400 cm−1 are shown in Fig. 4 and the spectrum for 3 is shown in Figure S7. To eliminate the interference peaks from bipyridine and phenanthroline, the IR spectra of previously reported complexes [VO(phen)2Cl]Cl·H2O77 and [VO2(bpy)2]Cl·4H2O78 were also re-recorded (Fig. 4). The spectra of 1 ~ 3 show well-resolved strong and sharp absorption bands for the carboxy groups of coordinated glycolates. For 1 and 2, the asymmetric stretching vibrations νas(CO2−) are observed at 1600 and 1607 cm−1 and the corresponding symmetric stretching vibrations νs(CO2−) appear at 1390 and 1377 cm−1 respectively. For 3, the asymmetric stretching vibrations νas(CO2−) are observed at 1655, 1649, 1630, 1597, 1561 and 1554 cm−1. The corresponding symmetric stretches νs(CO2−) appear at 1421, 1412, 1356 and 1317 cm−1. All of the carboxy absorptions shift to lower frequencies in comparison with those of free ligand H2glyc. The frequency differences ∆[νas(CO2−)-νs(CO2−)]79 are greater than 200 cm−1, which are consistent with the monodentate fashion that the carboxy group is coordinated to the metal ion, and are in agreement with the structural data observed with X-ray crystallography. The vibrational bands above 2000 cm−1 are assigned to C−H, N−H and O−H stretching modes. The features in the region of 920–990 cm−1 indicate the existence of V=O bond, and are consistent with the values observed in other vanadium complexes.38,41,43,80,81

Figure 4.

Figure 4.

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FT-IR spectra of [VO(Hglyc)(phen)(H2O)]Cl·2H2O (1), [VO(phen)2Cl]Cl·H2O, [VO(glyc)(bpy)(H2O)] (2) and [VO2(bpy)2]Cl·4H2O in the regions of 1800–400 cm−1.

The C–O stretching vibrations in alcohols produce bands in the region 1260–1000 cm−1.82 Based on the observations on some alcohols and α-hydroxycarboxylic acids listed in Table S7, the peaks of 1 at 1051.3 and 1039.5 cm−1 are assigned to the protonated C–OH stretching vibrations, while the peaks of 2 at higher frequencies 1074.4 and 1061.0 cm−1 are assigned to the deprotonated C–O stretching vibrations.65 All of the C–O(H) absorptions were shifted to lower frequencies in comparison with H2glyc at 1086 cm−1. The comparison of the IR spectra for 1, 2, [VO(phen)2Cl]Cl·H2O77 and [VO2(bpy)2]Cl·4H2O78 also supports the assignments for the peaks in Fig. 4. The protonated glycolato oxidovanadium complex 1 shows a red-shift (about 23 cm−1) in comparison with the deprotonated glycolato oxidovanadium complex 2. Besides, theoretical frequency calculations using Gaussian 09 for 1 and 2 (Figure S8) also exhibit the same red-shift trend, which show ν(C–O) and ν(C–OH) at 1098 and 1036 cm−1 respectively. This is the first time a similar pair of protonated and deprotonated complexes with the same V-homocitrate local coordination structure was investigated.

In Fig. S7 the C–O stretching vibrations of 3 are more complex due to the multiple types of coordination of glycolates. Complex 3 containing both α-alkoxy and α-hydroxy groups exhibits two absorptions at 1088.3 and 1064.5 cm−1, which are preliminarily assigned to the deprotonated C–Oα-alkoxy and protonated C–Oα-hydroxy stretching vibrations respectively. Therefore, complex 3 can serve as a mixed-(de)protonated model compound for the coexisting state of protonated and deprotonated homocitrates in nitrogenase.

Table 2 shows characteristic peaks of C–O or C–OH stretching vibrations of α-hydroxycarboxylato vanadium and molybdenum complexes, which serve as model compounds for FeV-co and FeMo-co. In analyzing these C–O/C–OH stretching vibrations, we have found that, whether in vanadium or molybdenum complexes, the protonated C–OH stretching vibrational frequencies are generally lower than the deprotonated C–O stretching vibrations. For example, previously reported lactato molybdenum(IV) complexes Na2[Mo3SO3(lact)3(im)3]·10H2O3 with α-alkoxy coordination and [MoIV3S4(PPh3)3(Hlact)2lact]67 with α-alkoxy/α-hydroxy coordinations also show red-shift from 1095.4, 1060.8, 1049.9 to 1090, 1036 cm−1 respectively. In brief, the ν(C–OH) of these complexes can be as low as 1030 cm−1, or even shifted to 1005 cm−1, while ν(C–O) are almost around 1080 cm−1. This is because the protonation weakens the strength of the C–O bond, resulting in the red-shift of vibrational frequency. As we can see in Table 2, the C−Oα-hydroxy distances (1.44av Å) of protonated compounds are subtly longer than C−Oα-alkoxy distances of deprotonated compounds (1.415av Å), and comparable with those of FeV-co (1.443av Å) and FeMo-co (1.449av Å). This is in accordance with the proposition of α-hydroxy coordination models in cofactors. The C–Oα-alkoxy vibrational frequencies of deprotonated homocitrate model complexes, [V2O3(phen)3(R,S-H2homocit)2(H2O)]Cl·6H2O41 and K2[MoVIO2(R,S-H2homocit)2]·2H2O61 were both observed to have an IR peak at 1084 cm−1. Although model complexes of protonated Mo/V homocitrates have not yet been obtained, the corresponding IR absorptions of the C−Oα-hydroxy bonds should shift to lower wave numbers according to the Hook law.

Table 2.

The C−O(H) bond distances (Å) and IR vibrational frequencies (cm−1) of C−Oα-alkoxy or C−Oα-hydroxy groups.

Samples C−O(H) distances (Å) Frequencies (cm−1)
Protonated
[VIVO(Hglyc)(phen)(H2O)]Cl·2H2O (1) 1.422(4) 1051.4m, 1039.5w
[VIVO(Hmal)(bpy)]·H2O34 1.433(4) 1085.3m, 1030.5m
[VIVO(S-Hcitmal)(bpy)]·2H2O34 1.444(5) 1059.6w, 1035.5m
[VIVO(H2cit)(bpy)]·2H2O34 1.440(8) 1068.7m, 1032.7s
(Et4N)2[Mo0(Hmal)(CO)3]68 (non-nature) 1.440(5) 1007w
(Et4N)3[Mo0(Hcit)(CO)3]68 (non-nature) 1.462(4) 1005m
(Et4N)2[Mo0(Hcitmal)(CO)3]68 (non-nature) 1.44(1) 1007w
Deprotonated
[VIVO(glyc)(bpy)(H2O)] (2) 1.412(3) 1074.4s, 1061.0m
K2[MoVIO2(glyc)2]·H2O53 1.404(5)av 1094s, 1082s, 1066s
[Mo3SO3(glyc)2(im)5]·im·H2O3 1.408(3)av 1099.0m, 1070.9s
{Na2[MoVIO2(S-lact)2]}3·13H2O53 1.427(8) 1085s, 1057s
Na2[Mo3SO3(R,S-lact)3(im)3]·10H2O3 1.404(4)av 1095.4m, 1060.8m, 1049.9m
[VIV/V2O3(phen)3(Hcit)2(phen)3O3V2]·12H2O41 1.410(2) 1073m
(NH4)4[(MoVIO3(cit)]·2H2O57 1.422(3) 1093m, 1073m
[(MoVO)2O(bpy)2(H2cit)2]·4H2O58 1.435(5)av 1078m
[(MoVO2)2(phen)(H2cit)(H2O)2]·H2O58 1.419(8) 1086m
[VIV/V2O3(phen)3 (R,S-H2homocit)2(H2O)]Cl·6H2O41 1.410(7) 1084m
K2[MoVIO2(R,S-H2homocit)2]·2H2O61 1.419(2)av 1083.8m
Protonated and deprotonated
(NH4)2[VIVO(Hglyc)2(glyc)]·H2O (3) 1.415(4)av 1088.3s, 1064.5s
[MoIV3S4(PPh3)3(Hlact)2lact]67 1.431(9)av 1090s, 1036s
 
FeV-co 1.443av1, 7 ---
FeMo-co 1.449av3 1084s, 1031s
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Re-evaluation of IR Spectrum of extracted FeMo-co.

To identify the protonation state of homocitrate in FeMo-co, we have re-examined the IR spectrum of FeMo-co extracted from nitrogenase purified from Azotobacter vinelandii (Fig. 5). The IR spectrum for the film sample of extracted FeMo-co exhibits absorption peaks at 1672, 1606, 1483, 1457, 1399, 1375, 1332, 1319, 1242, 1183, 1084, 1031, 1006, 984, 801, 748, 698, 647, 614 and 578 cm−1 respectively. Qualitative assignments of extracted FeMo-co are given in Table 3, where the vibrations of the following groups: COO−, C–C, C–O, Mo–O, tetraethylammonium chloride (TEAC) and N-methylformamide (NMF) are identified. The IR spectrum for TEAC is also shown in Fig. 5.

Figure 5.

Figure 5.

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FT-IR spectra of extracted FeMo-co and tetraethylammonium chloride (TEAC) in the region of 1800‒560 cm−1.

Table 3.

Qualitative assignments of the most intensive bands in IR spectrum of extracted FeMo-co.

Frequency assignments (cm−1)
Our work Orme-Johnson’s work9
1672 νas(CO2−)+
Partial NMF		 1664 NMF
1606 νas(CO2−) 1600 νas(CO2)
1483 TEAC
1457 TEAC
1399 νs(CO2−)+ Partial TEAC 1389 νs(CO2−)
1375 1357 νs(CO2−)
1332 TEAC
1319 -
1242 NMF
1183 ν(C–C)+
Partial TEAC		 1127 dithionite
1084 ν(C–O)
1031 ν(C–OH)
1006 TEAC 1002
984 -
801 TEAC 842
647 -
614 NMF
578 ν(Mo–O)
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The complexes’ C–O(H) stretching frequencies in Table 2 can serve as references for the C–O(H) vibrations in FeMo-co. The oxidation state of Mo atom in FeMo-co was assigned as Mo(IV)74 and Mo(III)75,76 previously, which are close to those of Mo(IV) complexes, such as Na2[MoIV3SO3(R,S-lact)3(im)3]·10H2O3 and [MoIV3S4(PPh3)3(Hlact)2lact].67 Therefore the C–OH or C–O vibrational frequency of FeMo-co is inferred to be around 1030 cm−1 or 1080 cm−1 respectively, which are analogous to the assignments of C–OH or C–O vibrations in complexes 1 and 2.

The peak at 1672 cm−1 in extracted FeMo-co IR is assigned to the asymmetric vibration νas(CO2−) of homocitrate, in comparison with 1675 cm−1 for K2[MoVIO2(R,S-H2homocit)2]·2H2O.61 It was assigned only to NMF in the previous reference.9 We therefore assign this peak to νas(CO2−) of extracted FeMo-co and the NMF absorptions.

The peak at 1606 cm−1 is assigned to the asymmetric vibration νas(CO2−) for homocitrate in extracted FeMo-co, corresponding to 1589 cm−1 for K2[MoVIO2(R,S-H2homocit)2]·2H2O61 and 1607 cm−1 for [V2O3(phen)3 (R,S-H2homocit)2(H2O)]Cl·6H2O41. This is consistent with the previous assignment by Orme-Johnson and coworkers9, but with a clearer resolution.

The peaks at 1399 and 1375 cm−1 for extracted FeMo-co should belong to the symmetric carboxyl vibrations, in comparison with the peaks 1390 cm−1 for K2[MoVIO2(R,S-H2homocit)2]·2H2O61 and 1383 and 1346 cm−1 for [V2O3(phen)3(R,S-H2homocit)2(H2O)]Cl·6H2O,41 which are consistent with Orme-Johnson’s observation.9 But the peaks in the range of 1493 ~ 1375 cm−1 also overlap with strong absorptions of TEAC, therefore 1399 and 1375 cm−1 could be assigned to νs(CO2−) of extracted FeMo-co and the TEAC absorptions.

The peak at 1183 cm−1 for extracted FeMo-co is assigned to C–C vibration, while the 578 cm−1 peak in the FeMo-co spectrum is assigned to Mo–O vibration in comparison with 553 and 540 cm−1 in K2[MoVIO2(R,S-H2homocit)2]·2H2O.61 The strong peaks at 1006 and 801 cm−1 are assigned to the vibrations of TEAC.

Most importantly, the peaks at 1083 and 1031 cm−1 in the IR-spectrum of extracted FeMo-co are assigned to C–O and C–OH vibrations as mentioned in the beginning of this section. The absorption at 1031 cm−1 is corresponding to the protonated C–OH at 1051, 1039 cm−1 in 1, 1032 cm−1 in [VIVO(H2cit)(bpy)]·2H2O,34 1036 cm−1 in [MoIV3S4(PPh3)3(Hlact)2(lact)]62 respectively. The peak at 1083 cm−1 is corresponding to the deprotonated model compounds [VIV/V2O3(phen)3(R,S-H2homocit)2(H2O)]Cl·6H2O41 (1084 cm−1) and K2[MoVIO2(R,S-H2homocit)2]·2H2O61 (1084 cm−1). Therefore, protonated and deprotonated homocitrates coexist in the particular extracted FeMo-co film evaluated here.

CONCLUSIONS

Here report a pair of mononuclear protonated and deprotonated glycolato oxidovanadium complexes with similar bidentate chelated local structure of V-homocitrate: [VO(Hglyc)(phen)(H2O)]Cl·2H2O (1) and [VO(glyc)(bpy)(H2O)] (2). The glycolate ligand coordinated bidentately to the central vanadium with protonated α-hydroxy and α-carboxy oxygen atoms in 1, while coordinated with deprotonated α-alkoxy and α-carboxy oxygen atoms in 2. In oxidovanadium triglycolate (NH4)2[VO(Hglyc)2(glyc)]·H2O (3), the glycolate ligands coordinated to vanadium through protonated α-hydroxy, deprotonated α-alkoxy and α-carboxy groups. The crystal structural data show that the elongated protonated V/Mo−Oα-hydroxy distances are close to those of FeV/Mo-cos, which indicate a proposed protonated α-hydroxy group in FeV/Mo-cos.

Based on the comparisons of C−Oα-alkoxy/α-hydroxy stretching vibrations of vanadium and molybdenum complexes, the C–OH stretching vibration will shift to a lower wave number in comparison with C–O stretching vibrations. The IR spectrum for extracted FeMo-co shows two absorption peaks at 1084 and 1031 cm−1, indicating a co-existence of deprotonated α-alkoxy and protonated α-hydroxy coordination in the Mo-homocitrate of FeMo-co. The IR spectra for some of the previously reported Mo/V complexes were also cited to compare with the three special oxidovanadium complexes in this publication and with the extracted FeMo-co. Nevertheless, the ≡C–O(H) peaks are assigned for the first time for all these samples.

EXPERIMENTAL SECTION

Materials and Instrumentation.

All solvents and reagents (in commercially analytical grade) were used without further purification. The pH value was determined by a PHB-8 digital pH meter. Elemental analyses (for C, H and N) were performed with a Vario EL III CHN elemental analyzer. Infrared spectra were measured in the range 400‒4000 cm−1 on a Nicolet FT-IR spectrometer with samples in KBr plates. The solid diffused UV/Vis spectra were recorded at 293K using a Cary 5000 UV-visible-NIR spectrophotometer in the 200‒800 nm range. Solid and solution (in DMSO) electron paramagnetic resonance (EPR) spectra were obtained by a Bruker EMX-10/12 spectrometer using crystalline samples at low temperatures. The FTIR of extracted FeMo-co was measured using Bruker V66/S and V70/v FTIR spectrometers at UCD and LBNL, with FeMo-co film on ZnSe or polyethylene plates respectively for different wave number regions. To minimize the NMF amount left in the FeMo-co film, the samples were pumped for more than 8 hrs before the measurement. Solution 13C NMR spectrum of 1 with long time superimposition was recorded on a Bruker Avance III 600MHz NMR spectrometer with D2O, using DSS (sodium 2,2-dimethyl-2-silapentane-5-sulfornate) as an internal reference.

Cell Growth and Purification of Nitrogenase Proteins.

The Aν wild-type strain was grown in the absence of a fixed-nitrogen source in a 24-L fermenter at 30 °C in a modified, liquid Burk medium.83 All cultures contained 20 µM FeCl3 and 10 µM Na2MoO4 and were grown to a final cell density of 250 Klett units recorded on a Klett–Summerson meter equipped with a number 54 filter. All manipulations of nitrogenase proteins were performed anaerobically using either a Schlenk line or an anaerobic glovebox operating at less than 1 ppm O2. After harvesting, cell extracts were prepared by diluting the whole cells with an equal amount of 50 mM Tris pH 8.0 buffer prior to passing through a French pressure cell and a centrifuge at 98000 g for 90 min. Nitrogenase component proteins were separated by anaerobic Q-Sepharose anion exchange column chromatography using a linear NaCl concentration gradient. Aν2 was purified to homogeneity by fractionation from a second Q-Sepharose column. Aν1 was further purified by Sephacryl S-200 gel filtration and phenyl-Sepharose hydrophobic-interaction chromatography.84 The purified Nitrogenase proteins were concentrated individually using an Amicon microfiltration pressure concentrator before buffer exchange to 25 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl2, and 2 mM Na2S2O4 by dialysis at 4 °C. Purified wild-type Aν1 had specific activities of 2200 nmol of H2 (min·mg·protein)−1 at 30 °C, when assayed in the presence of an optimal amount of the purified complementary component protein as described previously.84 Protein concentrations were determined by the Lowry method.

Extraction of FeMo-co from Aν1.

Aν1 was purified as above through the gel-filtration step, yielding protein with a specific activity of ~1000 nmol of H2 (min·mg protein)−1 and a Mo content of ~1 g·atom per mol of Aν1. After dialysis to lower the NaCl concentration, the Aν1 was loaded onto a DE-52 cellulose column that had been washed with 50 mM Tris pH 7.4 buffer containing 2 mM Na2S2O4. The bound protein was washed with N,N-dimethylformamide containing 50 mM 2,2′- bipyridine, 5 mM phosphate buffer pH 8, with 2 mM Na2S2O4, and water (ca. 5% v/v) until the noncofactor iron was completely eluted. The column was then washed with N-methylformamide (NMF) containing 5 mM phosphate buffer pH 8, with 2 mM Na2S2O4, and water (ca. 5% v/v), and FeMoco was then eluted with NMF that contained 500 mM tetraethylammonium chloride, 5 mM phosphate buffer pH 8, with 2 mM Na2S2O4, and water (ca. 5% v/v). The eluted FeMo-co was concentrated approximately 20-fold by distilling off the NMF under vacuum at 40 °C. FeMo-co was assayed85 by reconstitution of the DJ42 Aν strain, which has a deletion for the FeMo-co biosynthetic genes nifENX. The FeMo-co used in this study activated a DJ42 crude extract and produced 75 nmol of H2 (min·mg protein)−1.

X-Ray Crystallography.

The crystal structural data for 1 ~ 3 were collected on an Oxford Gemini CCD diffractometer, with graphite monochromatic Mo-Kα radiation (λ = 0.71073 Å) at 173 K. Muti-scan absorption corrections were applied. Direct methods structure solution, difference Fourier calculations, and full-matrix least-squares refinements against F2 were performed with SHELXL-2018/3 using the OLEX2 crystallographic software package86–88. All non-hydrogen atoms were refined anisotropically, while the hydrogen atoms of carbon atoms were generated geometrically and the hydrogen atoms of water molecules, ammonium ions and hydroxyl groups were located from differential Fourier maps and refined isotropically. To obtain reasonable structures, some “dfix” restraints were applied to the water molecules and hydroxyl group. The distances of O–H were restrained to be 0.85 Å: O1w–H1wA, O1w–H1wB, O2w–H1wA, O2w–H1wB, O3w–H1wA, O3w–H1wB and O1–H1 in 1, O1w–H1wA, O1w–H1wB in 2. The angles of H–O–H were restrained by fixing the distances of the two hydrogen atoms at 1.39 Å: H1wA…H1wB, H2wA…H2Wb and H3wA…H3wB in 1.

Computational method.

The geometry optimizations and frequency calculations were carried out using Gaussian 09.89 The molecular structures of [VO(Hglyc)(phen)(H2O)]+ cation of 1 and neutral molecule of 2 were optimized separately using DFT method. The 6–31G* (d, p) basis set was used for all atoms and B3LYP exchange–correlation functional was utilized to evaluate their performances in reproducing the solid-state structures and spectroscopic properties.

Synthesis of [VO(Hglyc)(phen)(H2O)]Cl·2H2O (1).

Vanadium pentoxide V2O5 (0.091 g, 0.50 mmol), glycolic acid in excess (0.60 g, 8.0 mmol), and 1,10-phenanthroline (0.10 g, 0.51 mmol) were dissolved in water (8 mL) with continuous stirring. The pH value was adjusted to 1.0 with dilute hydrochloric acid (1.0 M). The mixture was placed in a Teflon-lined stainless steel bomb. The bomb was heated to 443 K for 3 days and cooled with programmed control. The bluish green solution was evperated at room temperature for two months to grow blue crystals. The crystals were collected and washed with ethanol to afford 1 (0.067 g, 32% yield based on phen). Elemental analysis (calc. for C14H11ClN2O7V): C, 40.8; H, 4.2; N, 6.8%. Found: C, 40.6; H, 4.0; N, 6.4%. IR (cm−1): 3435(s), 3367(s), 3230(s), 3096(s), 3061(s), 2665(s), 2595(s), 2135(m), 2002(w), 1966(w), 1934(w), 1600(vs), 1522(s), 1493(m), 1426(s), 1390(s), 1341(m), 1303(m), 1220(w), 1211(w), 1200(w), 1151(w), 1141(w), 1109(m), 1051(m), 1041(w), 985(s), 940(m), 912(w), 875(m), 850(m), 799(w), 777(m), 742(m), 723(s), 654(m), 579(m), 559(m), 515(m), 500(m), 487(m), 435(m).

Synthesis of [VO(glyc)(bpy)(H2O)] (2).

Vanadium pentoxide V2O5 (0.091 g, 0.50 mmol), glycolic acid in excess (0.40 g, 5.26 mmol), and 2,2’-bipyridine (0.080 g, 0.50 mmol) were dissolved in water (8.0 mL) with continuous stirring. The pH value was adjusted to 3.0 with dilute potassium hydroxide (1.0 M). The bomb was heated to 443 K for 3 days and cooled with programmed control. The brown green crystals were collected and washed with ethanol to afford 2 (0.090 g, 56% yield based on bpy). Elemental analysis (calc for C12H12N2O5V): C, 45.7; H, 3.8; N, 8.9%. Found: C, 45.4; H, 3.6; N, 8.5%. IR (cm−1): 3435(m), 3111(m), 3100(m), 3057(m), 3033(m), 2863(m), 2829(m), 1607(vs), 1575(m), 1492(m), 1474(m), 1442(s), 1377(s), 1324(m), 1314(m), 1246(w), 1226(w), 1174(w), 1157(w), 1100(w), 1074(s), 1061(m), 1044(w), 1024(m), 1012(w), 968(s), 919(m), 769(s), 736(s), 653(m), 632(m), 596(m), 575(m), 520(m), 483(w), 443(w), 425(w).

Synthesis of (NH4)2[VO(Hglyc)2(glyc)]·H2O (3).24,25

Oxidovanadium sulfate (326 mg, 2.0 mmol) and glycolic acid (456 mg, 6.0 mmol) were dissolved in water (5.0 mL). The pH value of the solution was adjusted to 5.0 by concentrated ammonium hydroxide with continuous stirring. After one month, the light purple precipitate was collected and washed with water and ethanol to afford 3. (0.174g, 25% yield based on vanadium). Anal. Calc. for C6H18N2O11V: C, 20.9; H, 5.3; N, 8.1. Found: C, 20.8; H, 5.5; N, 8.0 (%). IR (cm−1): 3399(m), 3180(s), 3053(s), 2914(m), 2840(m), 1655(s), 1649(s), 1630(s), 1597(m), 1561(s), 1554(s), 1502(w), 1465(m), 1449(m), 1421(s), 1412(s), 1356(s), 1317(s), 1247(w), 1237(w), 1088(s), 1065(s), 1006(w), 955(s), 935(s), 740(m), 716(m), 603(m), 583(m), 568(m), 522(s), 483(w), 477(w), 444(m), 432(m), 419(m).

Supplementary Material

SI

ACKNOWLEDGMENT

We thank the supports from the National Science Foundation (21773196), the National Science Foundation of Fujian (2016I0101) and NIH GM-65440 (S.P.C.) for their generous financial supports. What’s more, we thank Ms. La-Jia Yu in EPR measurements.

ABBREVIATIONS

FeMo-co

FeMo-cofactor

H4homocit

homocitric acid

FeV-co

VFe-cofactor

phen

phenanthroline

H2glyc

glycollic acid

bpy

bipyridine

TEAC

tetraethylammonium chloride

Footnotes

ASSOCIATED CONTENT

Supporting Information

CCDC 1472398, 1472399 and 1570694 contain the supplementary crystallographic data for 1 ~ 3. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. It contains three parts of information: Part I Syntheses of [VO(phen)2Cl]Cl·H2O77 and [VO2(bpy)2]Cl·4H2O78, ORTEP plots, packing diagrams, UV-Vis, FT-IR, EPR, Crystallographic data, selected bond distances and angles, hydrogen bonds, valence bond analyses for complexes 1 ~ 3. The comparisons of V/Mo–O distances in α-hydroxycarboxylato vanadium/molybdenum complexes. Part II FT-IR spectrum of extracted FeMo-co. Part III IR spectra of all the compounds in Tables 2 and 3.

Wang-Tin Jin and Hongxin Wang are co-first authors. The authors declare no competing financial interest.

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