Diheme Cytochrome c: StructureâFunction Correlation and Effect of

Article Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

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Diheme Cytochrome c: Structure−Function Correlation and Effect of Heme−Heme Interactions Firoz Shah Tuglak Khan,† Sayantani Banerjee,† Devesh Kumar,*,‡ and Sankar Prasad Rath*,† †

Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur-208016, India Department of Physics, School for Physical and Decision Sciences, Babasaheb Bhimrao Ambedkar University, Lucknow-226025, India

‡

Inorg. Chem. Downloaded from pubs.acs.org by DURHAM UNIV on 08/30/18. For personal use only.

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ABSTRACT: We explore here the structure−function relationship of the diheme cytochrome c using synthetic diheme analogs which serve as a convenient tool to investigate various aspects of Nature’s sophisticated design in vitro. A large series of diiron ethane-bridged porphyrin dimers, both in the oxidized and the reduced states, are synthesized and their structural, chemical, and electrochemical properties have been scrutinized. Interestingly, the iron-to-iron nonbonding separation observed in such dihemes ranges from 9.49 to 10.06 Å which is very similar to the separation of 9.4 and 9.9 Å observed in the crystal structures of diheme cytochromes c isolated from Geobacter sulfurreducens and Haemophilus influenza, respectively. The FeIII/FeII redox couple in the diheme complex is shifted toward more positive than their monomeric analog. Present study unmasks the electronic structure and properties of diheme centers and also highlights the significance of their structural arrangement and axial ligand orientation, and heme-to-heme separation. The Atoms in Molecules (AIM) analysis suggests long-range attractive dispersion forces between the heme units for the observed structure and properties in dihemes.

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INTRODUCTION Multiheme cytochromes represent an extensive class of hemoproteins with consequential role in enzymatic catalysis and electron transport in biology.1−4 The majority of them belong to the family of cytochromes c in which the heme units are covalently connected to the polypeptide chain via formation of two thioether bonds between two cysteine residues and the vinyl side-chains of the porphyrin ring.1−5 A low amino acid residue to heme ratio and bis-histidine coordination, with few exceptions, characterizes them. Understanding the structural arrangement of multiheme units is crucial to gain more insights into the highly optimized properties of these multiheme cytochromes c. However, their spectroscopic investigation is often complicated due to the presence of large number of heme centers along with substantial intermacrocyclic coupling in between.1−4 However, several closely packed diheme blocks have been found in the structural arrangements of multiheme proteins.5 Interestingly, the array of such diheme motifs generates large and almost super imposable superheme motifs.1−3 Diheme cytochrome c (DHC2), found in the bacteria Geobacter sulfurreducens, is the simplest member of the multiheme family which has two spectroscopically inequivalent heme units having different redox properties.6a Stereoviews of the two diheme cytochromes are shown in Figure 1 in which diheme centers are also highlighted. Iron-to-iron nonbonding separa© XXXX American Chemical Society

tion of 9.4 Å has been observed in DHC2 which, however, results in an efficient coupling between the heme centers. An interplanar angle of 36° has been observed between two axially coordinated histidine ligands in heme unit I, whereas two axial ligands are nearly coplanar in heme unit II. Moreover, the porphyrin macrocycle of heme group I is almost planar whereas it is highly distorted in heme group II. Such differences in the porphyrin ring deformations as well as axial ligand orientations between the heme units in DHC26a have been proposed to be caused by interheme interactions. Heme arrangement similar to DHC2 has also been observed in the small subunit (NapB) of the periplasmic nitrate reductase occurring in Haemophilus influenza.6b In NapB, the two heme groups are oriented almost parallel to each other with dihedral angles of 11° and 21° between the two axially ligated imidazoles in the two heme groups. Iron-to-iron separation of 9.9 Å has been observed here which is also similar to the split-Soret diheme cytochrome c found in Desulfovibrio desulf uricans ATCC 27774.6c The above state of affairs has prompted us to investigate the structure−function correlation for diheme cytochrome c and the effect of the interheme interactions therein. The separation mediated sharp decrease in intramolecular interactions makes Received: May 18, 2018

A

DOI: 10.1021/acs.inorgchem.8b01368 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

1, converts immediately into 1·X2 in quantitative yields (Scheme 1).8 Scheme 1. Synthetic Outline

Figure 1. Relative stereoviews of (A) diheme cytochrome c isolated from G. sulf urreducens (PDB code 2CZS)6a and (B) split-Soret cytochrome c isolated from D. desulf uricans (PDB code 1H21).6c

However, addition of axial ligand L into the dichloromethane solution of 1·X2 (X: ClO4, Cl and TNP) results in the formation of the six-coordinate complex [1·(L)4](X)2 which has been isolated and structurally characterized. The axial ligands (L) that are used in the present investigation are imidazole (Im), 1-methylimidazole (1-MeIm), 2-methylimidazole (2-MeIm), 4-methylimidazole (4-MeIm), pyridine (Py), 3-cyanopyridine (3-CNPy), and 4-cyanopyridine (4-CNPy). Moreover, upon standing with excess L, the complex [1· (L)4](ClO4)2 spontaneously autoreduces13 into air-stable 2· (L)4 in quantitative yield. During such a process, large changes in the UV−visible spectra are observed. For example, gradual addition of imidazole to 1·(ClO4)2 produces a large spectral change in which peaks at 390 and 515 nm corresponding to 1· (ClO4)2 transformed into peaks at 418 and 549 nm due to the formation of [1·(Im)4](ClO4)2; Figure 2 demonstrates such spectral changes in the UV−visible region. Similar changes are also obtained with other imidazoles/pyridines as axial ligands. Figure 2 (trace B) compares the absorption spectra between [1·(4-MeIm)4](ClO4)2 and 2·(4-MeIm)4 in dichloromethane. Scheme 1 shows the synthetic outline and list of the complexes reported here along with their abbreviations used. The UV−vis spectra of the diheme complexes reported here are similar to the native diheme cytochrome c (DHC2) in their oxidized and reduced states. For example, the Soret and Qbands of [1·(4-MeIm)4](ClO4)2 are observed at 408 and 539 nm while in the reduced complex 2·(4-MeIm)4, the bands are, respectively, at 419 and 551 nm. It is, however, interesting to note here that the oxidized form of the diheme cytochrome c, DHC2, from G. sulfurreducens shows absorption maxima at 408 and 530 nm whereas the reduced form exhibits the same maxima at 419 nm (Soret) and 523, 552 nm.6a As one can see, electronic spectra of the synthetic diheme complexes are nearly identical with the DHC2 both in the oxidized and reduced forms. Crystallographic Characterizations. Dark red crystals of [1·(1-MeIm)4](ClO4)2 were grown by slow diffusion of nhexane into the benzene solutions of 1·(ClO4)2 containing 1methylimidazole in 1:5 molar ratio at room temperature in air. The complex crystallizes in the triclinic crystal system with the P1̅ space group. Two perspective views of the molecule are shown in Figure 3. Similarly, the crystals of [1·(2-MeIm)4]Cl2, [1·(4-CNPy)4](ClO4)2 and [1·(Py)4](TNP)2 were grown. In all the complexes, the iron centers are in a six-coordinate

close proximity of the heme units susceptible to significant modification of their intrinsic properties.3d,4b However, the study of structure−function activity relationships of such diheme cytochromes c are complicated by the presence of large polypeptide chains. Synthetic metalloporphyrins, therefore, serve as convenient systems for testing mechanistic hypotheses of enzymatic processes as they are unhindered by the absence of any associated biological superstructure. Active site analogs of such diheme cytochromes provide a tool to investigate various aspects of Nature’s sophisticated design in vitro which cannot be obtained through the native enzyme. We have been engaged in exploiting the significance and outcome of the heme−heme interactions using metalloporphyrin dimers.7−12 In the present work, we explore the structure−function relationship of the diheme cytochrome c using a series of synthetic diheme analogs. For that, covalently linked porphyrin dimer with highly flexible ethane bridge has been employed as a diheme analog in which two-heme units orient in an anti-conformation. A large series of such analogs, both in the oxidized as well as in the reduced states, are synthesized and their structural, chemical, and electrochemical properties have been scrutinized. Interestingly, the iron-to-iron nonbonding separation observed in such dihemes ranges from 9.49 to 10.06 Å (vide inf ra) which is very similar to the iron-toiron separation of 9.4 and 9.9 Å observed in the crystal structures of diheme cytochromes c isolated from G. sulfurreducens and H. influenza, respectively.6 Such similar iron-to-iron separation as well as relative structural arrangement of diheme units make the diiron ethane-bridged porphyrin dimer an excellent synthetic analog for exploring several aspects of Nature’s sophisticated design.

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RESULTS AND DISCUSSION μ-Oxo-syn-1,2-bis[5-(2,3,7,8,12,13,17,18 octaethylporphyrinato)iron(III)]ethane, 1, was synthesized using a procedure reported earlier.10a UV−vis spectroscopic data of 1 shows a Soret band at 399 nm and a Q-band at 579 nm in dichloromethane in which two porphyrin macrocycles are in face- to-face orientation. Upon addition of excess acid (HX) such as HCl, HClO4 and 2,4,6-trinitrophenol (HTNP), B


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Inorganic Chemistry

the narrow range of 1.948 to 1.974 Å while Fe−Nax distances are within 1.961−2.101 Å. These values are in the range of sixcoordinate low-spin iron(III) porphyrins reported earlier.13−18 The X-ray structures of [1·(1-MeIm)4](ClO4)2, [1·(2MeIm)4]Cl2, [1·(Py)4](TNP)2, and [1·(4-CNPy)4](ClO4)2 demonstrate nearly perpendicular axial ligand orientations in the distorted macrocyclic environments. Dark brown crystals of 2·(L)4 were grown at room temperature in air by slow diffusion of acetonitrile into the chloroform solution of 1·(ClO4)2 containing large excess of pyridine, 3-cyanopyridine and 4-cyanopyridine yielding 2· (Py)4, 2·(3-CNPy)4, and 2·(4-CNPy)4, respectively. All the complexes crystallize in triclinic crystal system having the P1̅ space group. A perspective view of 2·(Py)4 is shown in Figure 4 (trace B) while the perspective views and molecular packing diagrams of 2·(3-CNPy)4 and 2·(4-CNPy)4 are shown in Figures S9−S13. Here also, the axial ligands are in perpendicular orientations. Interestingly, iron-to-iron nonbonding distances along with other structural and geometrical parameters of these synthetic dihemes are very similar to the naturally occurring DHC2 as summarized in Table 1. The selected bond distances and angles are listed in Table S1 while the crystal data and data collection parameters are listed in Table S3. As observed from Table 1, Fe−Npor distances are in the narrow range of 1.988 to 1.991 Å while Fe−Nax distances are within 1.972 to 2.003 Å. These values are in the range of low spin iron(II) porphyrins observed in the literature.13−18 As can be seen in Table 1, iron(III) complexes are more distorted compared to their iron(II) analogs and the out-ofplane displacements of the porphyrin core atoms of 2·(L)4 and[1·(L)4]X2 are compared in Figures 5 and S14. One reason for such a decrease in the ring deformation on going from iron(III) to iron(II) is the difference in the ionic radii of iron. It is interesting to compare the porphyrin ring deformations in [1·(Py)4](TNP)2, 2·(Py)4, and their monomeric iron(II) analog Fe(OEP)(Py)2 (Tables S4 and S5), which, however, produces a nearly planar porphyrin ring. It has been observed that the macrocycles in the porphyrin dimers are much more distorted as compared to their monomeric analog. In a nonplanar porphyrinic environment, the axial ligand orienta-

Figure 2. (A) Time resolved UV−vis spectral changes upon gradual addition of imidazole up to 6 equiv to a solution of 1·(ClO4)2 in dichloromethane at 295 K. Arrows indicate an increase or decrease of band intensity. (B) UV−vis spectra (in dichloromethane at 295 K) comparing [1·(4-MeIm)4](ClO4)2 (red line) and 2·(4-MeIm)4 (green line).

octahedral geometry. The complex [1·(2-MeIm)4]Cl2 crystallizes in the monoclinic crystal system with the P21/n space group while [1·(4-CNPy)4](ClO4)2 crystallizes in triclinic and [1·(Py)4](TNP)2 in monoclinic crystal system having space groups of P1̅ and P21/c, respectively. Perspective views and molecular packing diagrams of the complexes are given in Figures S1−S7 and the out-of-plane displacement plots comparing the ring deformations are given in Figure S8. The selected bond distances and angles are given in Table S1 while the crystal data and data collection parameters are given in Table S2. As observed from Table S1, Fe−Npor distances are in

Figure 3. (A) Perspective views of [1·(1-MeIm)4](ClO4)2 (A, side view and B, top view without counteranions) and [1·(2-MeIm)4]Cl2 (C, side view and D, top view without counteranions) showing 50% thermal contours for all the non-hydrogen atoms at 100 K (hydrogen atoms have been omitted for clarity). C


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Inorganic Chemistry

Figure 5. Out-of-plane displacement (in units of 0.01 Å) of the porphyrin core atoms of [1·(Py)4](TNP)2 (black line) and 2·(Py)4 (red line) from the least-squares plane of the C20N4 porphyrinato core. The horizontal axis represents the bond connectivity between atoms.

tions have been demonstrated to switch between perpendicular and parallel upon changing the oxidation state of iron,13 however, no such change is observed in the diheme complexes reported here. Moreover, in case of Fe(OEP)(Py)2 the axial ligands are found to be in parallel orientation, whereas in its diheme analog 2·(Py)4, it was found to be perpendicular. The heme−heme interaction in diheme has increased the porphyrin ring deformations, and subsequently, the perpendicular alignment of axial ligands are stabilized in both diiron(III) and diiron(II) porphyrin dimers reported here.

Figure 4. Perspective views of (A) [1·(Py)4](TNP)2, (B) 2·(Py)4, and (C) Fe(OEP)(Py)2 showing 50% thermal contours for all the non-hydrogen atoms at 100 K (hydrogen atoms have been omitted for clarity).

Table 1. Selected Structural and Geometrical Parameters of [1·(L)4](X)2, 2·(L)4, and Fe(OEP)(Py)2 along with the Native Diheme Cytochrome c Fe−Npor (Å)a

Complex [1·(1-MeIm)4](ClO4)2

1.974(2)

[1·(2-MeIm)4]Cl2

1.973(2)

[1·(Py)4](TNP)2

1.956(2)

[1·(4-CNPy)4](ClO4)2

core-I

1.959(4)

core-II

1.948(4)

2·(4-CNPy)4

1.991(3)

2·(3-CNPy)4

1.9888(18)

2·(Py)4

1.988(3)

Fe(OEP)(Py)2 DHC2 (isolated from G. sulfurreducens)

DHC2 (isolated from H. influenza)

DHC2 (isolated from D. desulf uricans)

core-I

2.001(2) 2.044

core-II

2.033

core-I

1.982

core-II

1.996

core-I

1.942

core-II

1.976

Fe−Nax (Å)

Φ (deg)b

θ (deg)c

Δ24 (Å)d

Fe···Fe (Å)

ref

1.961(2) 1.982(2) 1.991(4) 1.999(4) 1.986(2) 2.101(2) 1.991(3) 1.996(3) 1.998(3) 2.003(4) 1.972(3) 2.003(3) 1.9840(19) 1.9977(18) 1.990(3) 2.003(3) 2.048(2) 1.935 1.979 2.007 2.032 2.007 2.051 2.003 2.073 1.970 2.037 2.000 2.019

37.7

85.2

0.25

9.89

this work

43.5

87.6

0.26

9.91

this work

42.2

87.6

0.31

9.49

this work

42.7

87.2

0.28

9.67

this work

39.7

81.9

0.36

42.7

88.2

0.17

9.86

this work

38.1

81.8

0.20

10.06

this work

44.1

87.4

0.19

9.94

this work

32.1 44.1

0.0 36

0.02 0.07

9.38

this work 6a

30.0

2

0.21

42.6

21

0.13

9.90

6b

44.4

14

0.14

33.7

74

0.22

9.17

6c

38.6

23

0.15

Average value. bDihedral angle between the plane of the closest Npor−Fe−Nax and the axial ligand plane. cDihedral angle between two axial ligands. dAverage displacement of atoms from the least-squares plane of the C20N4 porphyrinato core.

a

D


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Inorganic Chemistry Mössbauer Spectroscopy. Mössbauer parameters are one of the most important tools to determine the spin states of iron(III) porphyrins.17,18 The spectra of the polycrystalline samples of [1·(L)4](X)2 (X = ClO4 and Cl) were recorded in zero magnetic field at 295 K and are shown in Figure 6. All the

Figure 7. X-band EPR spectra of (A) [1·(Im)4](ClO4)2, (B) [1·(4MeIm)4](ClO4)2 and (C) [1·(1-MeIm)4](ClO4)2 in dichloromethane at 77 K.

Figure 6. Zero-field Mössbauer spectra of the polycrystalline samples of (A) [1·(1-MeIm)4](ClO4)2 and (B) [1·(Im)4](ClO4)2 at 295 K.

1

H NMR Spectroscopy. The solution structure and properties of the complexes can be analyzed through their 1 H NMR spectra. The 1H NMR spectrum for [1·(Py)4](TNP)2 has been recorded in pyridine-d5. Eight methylene signals are observed at a region between 6.1 and 11.3 ppm while the bridging protons are further downfield shifted to 18.3 ppm. The meso protons are upfield shifted and observed at 0.8 and −5.9 ppm in a 2:1 intensity ratio (Figure 8). Similar peak

complexes show only one quadrupole doublet: for [1· (Im)4](ClO4)2, δ (ΔEQ) = 0.19 (2.12) mm s−1; [1·(1MeIm)4](ClO4)2, δ (ΔEQ) = 0.18 (2.19) mm s−1; [1·(2MeIm)4]Cl2, δ (ΔEQ) = 0.16 (1.55) mm s−1. Similarly, one quadrupole doublet was observed for [1·(Py)4](TNP)2 having δ (ΔEQ) = 0.15 (2.29) mm s−1. These values are characteristic of the iron(III) low-spin complex.17,18 Therefore, Mössbauer parameters are in line with the results obtained from the X-ray structures of the complexes (vide supra). EPR Spectroscopy. X-band EPR spectra were measured for [1·(L)4](ClO4)2 at 77 K which show rhombic type spectral features both in solid and solution phases. The spectra for the representative complexes in dichloromethane are shown in Figure 7. Upon simulations, g values that are obtained are as follows: g3 = 2.70, g2 = 2.27, and g1 = 1.75 for [1· (Im)4](ClO4)2; g3 = 2.67, g2 = 2.26, and g1 = 1.75 for [1·(4MeIm)4](ClO4)2; g3 = 2.86, g2 = 2.25, and g1 = 1.53 for [1·(1MeIm)4](ClO4)2. The spectra are of typical rhombic type that are usually observed for low-spin (S = 1/2) iron(III) porphyrins with two axially ligated imidazoles. It is interesting to note here that the X-band EPR of DHC2 also exhibits similar rhombic type spectra.6a However, with 4-fold symmetric porphyrins, the appearance of a rhombic spectrum for a low-spin iron(III) porphyrin usually indicates the two axial ligands lying in a common plane.13−16 The low-spin iron(III) porphyrins with perpendicularly oriented planar axial ligand usually display “large gmax” EPR spectra that mark the near degeneracy of the dxz and dyz orbitals.13−16 However, in the diheme complexes reported here, the intrinsic asymmetry in the porphyrin dimer lowers the symmetry leading to rhombic type EPR spectra, even though the planar axial ligands are in perpendicular orientations. Similar rhombic-type EPR data were also observed for the meso-hydroxy heme in which two axial imidazoles are in perpendicular orientation.16a

Figure 8. 1H NMR spectrum (in pyridine-d5 at 295 K) of [1· (Py)4](TNP)2. Peaks marked as “s” correspond to signals from solvent.

patterns have also been observed in the 1H NMR spectra of [1· (Im)4](ClO4)2, recorded in CDCl3, where eight methylene signals at 2.9(2), 4.6, 5.3, 6.0, 6.2, 6.4, and 6.9 ppm, bridging CH2 protons at 15.9, and two meso signals at −1.9 and −8.5 ppm in intensity ratio 2:1 are observed. Mulliken spin density calculation of [1·(Py)4]2+ (Figure S15) shows positive spin densities at the meso carbons while negative spins at the bridging carbons, thus, ascertaining the upfield and downfield shifting of corresponding proton signals as observed in their 1H NMR spectrum. The observed 1H NMR spectral pattern is of a typical hexacoordinated iron(III) low-spin complex.14,16−19 E


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Inorganic Chemistry The 1H NMR spectra of [1·(L)4](X)2 (X = ClO4 and Cl), where L are substituted imidazoles, have also been recorded and it was observed that the proton signals from imidazoles are shifted to both downfield and upfield regions which indicate πspin delocalization from iron(III) center to axially coordinated imidazole. In order to assign the proton peaks of the coordinated imidazole species, several substituted imidazoles have been used here. The 1H NMR spectra of the complexes are compared in Figure 9. The Ha proton of coordinated

Figure 10. Portion of cyclic voltammograms of (A) [1·(4-MeIm)4](ClO4)2, and (B) [1·(2-MeIm)4]Cl2 in CH2Cl2 (scan rate 100 mV/s) with 0.1 M tetra(n-butyl)ammonium hexafluorophosphate as supporting electrolyte. The reference electrode was Ag/AgCl.

were also carried out under identical conditions and the first oxidation couples for [Fe(OEP)(Im)2]ClO4, [Fe(OEP)(4MeIm)2]ClO4, [Fe(OEP)(1-MeIm)2]ClO4, and [Fe(OEP)(2MeIm)2]Cl are observed at 0.83, 0.84, 0.85, and 0.86 V, respectively. Thus, the first oxidation potential of the diheme species occurs at a potential much lower than its monomeric analog. The reduction of diheme complex [1·(L)4](X)2 has also been performed and the E1/2 of FeIII/FeII redox couple are observed at −0.27, −0.36, −0.37, and −0.42 V vs. Ag/AgCl for L= Im, 4-MeIm, 1-MeIm, and 2-MeIm, respectively, and three representative cyclic voltammograms are shown in Figure 11.

Figure 9. 1H NMR spectra (in CDCl3 at 295 K) of (A) [1· (Im)4](ClO4)2, (B) [1·(2-MeIm)4]Cl2, (C) [1·(1-MeIm)4](ClO4)2, and (D) [1·(4-MeIm)4](ClO4)2. Peaks marked as “x” belong to proton signals from free ligands.

imidazole in [1·(Im)4](ClO4)2 shifts to the upfield regions at −9.9 whereas the Hb and Hc protons shift downfield to 13.7 and 10.7 ppm, respectively. Mulliken spin density calculation on [1·(2-MeIm)4]Cl2 (Figure S16) displays considerable spin delocalization to the axially coordinated imidazole also. The spin densities observed is negative at Cb position while positive at Ca position. Therefore, the Cb protons should be downfield shifted while Ca proton is to be upfield shifted which is indeed observed in the 1H NMR spectra of the complex. Electrochemistry. Cyclic voltammetric experiments were done at 295 K under nitrogen in dichloromethane using 0.1 M tetra(n-butyl)ammonium hexafluorophosphate (TBAH) as the supporting electrolyte. The first oxidation couples for [1· (Im) 4 ](ClO 4 ) 2 , [1·(4-MeIm) 4 ](ClO 4 ) 2 , [1·(1-MeIm) 4 ](ClO4)2, and [1·(2-MeIm)4]Cl2 are observed at 0.60, 0.61, 0.62, and 0.64 V vs Ag/AgCl, respectively, which correspond to porphyrin-centered oxidation and two representative cyclic voltammograms are shown in Figure 10. Similarly, the electrochemical measurements for the monomeric analogs

Figure 11. Portion of cyclic voltammograms of (A) [1·(2-MeIm)4]Cl2, (B) [1·(4-MeIm)4](ClO4)2, and (C) [1·(Im)4](ClO4)2 in CH2Cl2 (scan rate 100 mV/s) with 0.1 M tetra(n-butyl)ammonium hexafluorophosphate as supporting electrolyte. The reference electrode was Ag/AgCl.

Coulometric reduction of [1·(L)4](ClO4)2 in dichloromethane at a constant potential has also been done and the progress of the reaction was monitored continuously by UV−vis spectroscopy as shown in Figure S17 for [1·(4-MeIm)4](ClO4)2, as a representative case. A gradual decrease of the Soret band characteristics of iron(III) porphyrin and the appearance of a new Soret band corresponding to iron(II) confirms the corresponding potential as that of the iron(III)/iron(II) redox couple. The cyclic voltammogram for the monomeric analog has also been recorded: the E1/2 of FeIII/FeII redox F


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Inorganic Chemistry couple for [Fe(OEP)(Im)2]ClO4, [Fe(OEP)(4-MeIm)2]ClO4, [Fe(OEP)(1-MeIm)2]ClO4, and [Fe(OEP)(2-MeIm)2]Cl are observed at −0.41, −0.42, −0.43, and −0.45 V, respectively. This observation demonstrates that not only the oxidation but also the reduction become easier in the diheme complexes as compared to their monoheme analogs. The plots of first oxidation potential and FeIII/FeII redox couple of diheme complexes vs pKa of the axial ligands (i.e., Im, 4-MeIm, 1MeIm, and 2-MeIm) are shown in Figure 12 in which both

Figure 13. uB3LYP/BS1 optimized geometry of [1·(2-MeIm)4]2+ with bond lengths in Å and angles in degrees. Parentheses contain the experimental value obtained from the X-ray structure of [1·(2MeIm)4]Cl2 (hydrogen atoms have been omitted for clarity).

experiment. For example, the optimized geometry of [1·(1MeIm)4](ClO4)2 displays angles of 94.3° and 85.4° between the two axial ligands in cores I and II, respectively, which are, however, close to the values observed in the X-ray structure of the complex. To gain more insights into the stabilization of such perpendicular orientation, 1D potential energy surface (PES) scan on [1·(1-MeIm)4](ClO4)2 has also been performed. One of the axial ligand from each of the cores was rotated in steps of 10° keeping other two axial ligands intact in their positions. The relative energy of the complex was then plotted against the dihedral angle as shown in Figure 14 (trace A). The lowest energy was observed when the dihedral angle between the axial ligands is ∼90° which is also close to the angle observed in the X-ray structure of the complex. Similar calculations are also

Figure 12. Plots of pKa20 of axial ligands vs (A) first oxidation, and (B) FeIII/FeII reduction potential (in V) of [1·(L)4](X)2 and [Fe(OEP)(L)2]X (X = ClO4,Cl). Red and blue lines correspond to diiron(III) porphyrin dimer and its corresponding monomeric analog, respectively.

diheme and monoheme complexes show a linear relationship. To gain more insights into such electronic effect in the diheme species, a series of theoretical calculations are performed (vide infra). Molecular Modeling. Density functional calculations have been carried out for several six-coordinate low-spin diiron(III) and diiron(II) porphyrin dimers in which the atom coordinates are taken directly from their single crystal X-ray structures. The geometries of [1·(L)4]2+, 2·(L)4, [FeIII(OEP)(L)2]+ and FeII(OEP)(L)2 (where L= Im, 2-MeIm, 1-MeIm and Py) are optimized and the calculations virtually reproduce the geometrical parameters observed in their X-ray structures (Figures 13 and S18−S22). Computational studies were done employing the Gaussian 09, revision B.01 package21 with unrestricted hybrid B3LYP22 exchange-correlation functionals and the basis set combination, termed as BS1, used is LANL2DZ (for Fe) and 6-31G** (for C, H, N, Cl and O). Upon going from diiron(III) to diiron(II), some structural changes are observed in the optimized structures as obtained in the experiment also (vide infra). The Fe−Npor bond distance elongates from 1.982 to 2.023 Å (for L = Py, as a representative case) which results in porphyrin core expansion. The intramolecular iron-to-iron distance increases on going from diiron(III) to diiron(II) which was also observed experimentally. Moreover, the optimized structures reproduce the perpendicular axial ligand orientation as observed in the

Figure 14. Plots showing the relative energy (ΔE) upon changing dihedral angles between the two 1-methylimidazole axial ligands for (A) [1·(1-MeIm)4](ClO4)2 and (B) 2·(1-MeIm)4. G


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Inorganic Chemistry

stable geometry was observed when the iron-to-iron separation was 10.05 Å which is in close agreement with the experiment. Upon varying the interheme separation, by increasing or decreasing the Fe···Fe distance, the molecule was more and more destabilized as evident from the relative energy curve (Figure 15). Eventually, at a distance beyond 11.0 Å, the curve

done on the uB3LYP/BS1 optimized geometry of its iron(II) analog 2·(1-MeIm)4; the relative energy plot with respect to dihedral angle also shows the stabilization of geometry having perpendicular axial ligand orientations only (Figure 14, trace B). The calculation on the optimized geometry of [1·(Py)4]2+ (without counteranions) also displays the perpendicular axial ligand orientation being stabilized by 36.9 kcal/mol over the parallel one. To understand more about the stability of the perpendicular orientation of the axial ligands, several singlepoint calculations are done on the optimized geometries of [1· (Im)4]2+ and 2·(Im)4 after removing ethyl substituents and the ethane bridge keeping everything else intact in the complexes. While perpendicular orientations in [1·(Im)4]2+ and 2·(Im)4 are stabilized over parallel by 13.5 and 3.8 kcal/mol, respectively, the energy differences, however, have reduced to 8.1 and 1.4 kcal/mol, respectively, after removal of ethyl substituents as well as the ethane bridge (Table S6). Interestingly, no change in the axial ligand orientation has been observed on going from diiron(III) to diiron(II) state; i.e., both the complexes were stabilized with a perpendicular orientation of the axial ligands. In contrast, axial ligand orientation has been found to vary between parallel and perpendicular just upon changing iron oxidation states from +3 to +2 in saddle-distorted iron porphyrins.13 This further justify the judicious choice of diheme over monohemes where the reorganization energy, on going from Fe(III) to Fe(II), has been minimized in diheme as there is negligible structural change with identical axial ligand orientation. To gain more insights into this reduction of reorganization energy, two monoheme complexes were optimized, in Fe(III) as well as Fe(II) state, having meso-methyl OEP core with imidazole and 2-methylimidazole axial ligands. Such a porphyrin core was chosen as it structurally resembled one-half of its porphyrin dimer. Fe(III) to Fe(II) reductions in diheme complexes having imidazole and 2-methylimidazole as axial ligands are found to be stabilized by 7.8 and 6.6 kcal/mol, respectively, over their monomeric analogs. This justifies the reduction in structural reorganization energy in dihemes over monohemes. The selected structural and geometrical parameters for the optimized geometries of [1·(L)4]2+ and 2·(L)4 (L= Py, Im, 1MeIm, and 2-MeIm) are compared in Table S7 and are also shown in Figures S18−S22. It has been experimentally found that the Fe(III) to Fe(II) reduction is much easier in dihemes as compared to their monoheme analogs (vide supra) which has also been reproduced by theoretical calculations. The redox potentials of [1·(Im)4]2+ and [1·(2-MeIm)4]2+ were calculated using a method reported earlier.23 The calculated FeIII/FeII redox potential was −0.06 V in diheme complex [1·(2-MeIm)4]2+ whereas it was −0.19 V in the monoheme [Fe(OEP)(2MeIm)2]+. Similarly, the redox potential for [1·(Im)4]2+ was 0.04 V which was −0.11 V in [Fe(OEP)(Im)2]+ (Table S8). This is also in line with the decrease of reorganization energy in diheme as compared to the monoheme analog which results in an easier reduction of diheme as compared to the monoheme. To gain more insights into the effect of heme−heme interactions in diheme, a series of DFT calculations were performed on [1·(Im)4]2+ by varying iron-to-iron separation from 9.5 to 11.0 Å in the increments of 0.1 Å in each step (Tables S9 and S10) and subsequently optimizing the geometry. This variation alters the interheme interaction between the heme units in the porphyrin dimer. The most

Figure 15. Plots of (A) relative energy (ΔE) of optimized geometries with Fe···Fe separation, (B) distance dependence of the pairwise interactions between the heme centers and (C) relative energy (ΔE) upon changing dihedral angle between two heme centers of [1· (Im)4]2+.

becomes almost parallel with respect to the X-axis, which indicates a negligible change in energy. This signifies that the diheme complex behaves as two separate monohemes at that distance. This gain in stabilization energy facilitates faster reduction of the iron(III) centers as compared to its monoheme counterpart which also closely agrees with the experiment. The redox potential for FeIII/FeII redox couple of [1·(Im)4]2+ at a Fe···Fe separation of 11.0 Å has also been calculated to be −0.22 V. However, when two hemes are brought closer, the redox potential for FeIII/FeII couple gradually decreases which has been found to be only 0.04 V at a iron-to-iron separation of 10.05 Å. Not only the iron-to-iron separations in dihemes but the spatial arrangement might also be responsible in modulating the redox property of the diheme. To explore such effect on the heme-to-heme relative orientation, a sequence of calculations were done on [1·(Im)4]2+ varying the relative dihedral angle (θ′) between two heme units from 0° (found as most stable orientation), in steps of 10° in either directions until the dihedral angle of 50° and the molecules are H


Article

Inorganic Chemistry subsequently optimized with fixed θ’ (Tables S11 and S12). A plot of relative energy of the optimized geometries vs θ′ has been shown in Figure 15 (trace C). The redox potentials of FeIII/FeII couples for [1·(Im)4]2+ were calculated to be −0.12 and 0.04 V, respectively, for 50° and 0° dihedral angles (θ′). This suggests that not only the iron-to-iron separation but also the relative heme orientations are also responsible for the change in their FeIII/FeII redox couple. Such structurally mediated heme−heme interactions thus serve as an efficient tool to modulate the redox potentials in dihemes. To understand the interaction between the two-heme units, AIM (atoms in molecules) analysis was executed. The software DAMQT 2.1.024 was utilized for the topographical analysis of uB3LYP/BS1 optimized geometry of [1·(Im)4]2+ and subsequent mapping shows several critical points between the two heme units (Figure 16, trace A). Analysis of such points

of L, quantitatively auto reduces into air-stable diiron(II) porphyrin dimer 2·(L)4. Such autoreduction produces large changes in the UV−vis spectra of the dihemes. Also, the spectral changes are similar as observed in the native diheme cytochromes c (DHC2) in their oxidized and reduced states. The X-ray structures of seven such dihemes along with a monoheme analog have been determined successfully which demonstrate low-spin state of iron. Structurally these diheme analogs closely resemble with the naturally occurring DHC2. The solid-state structures are also intact in solution which is further supported by 1H NMR and EPR studies. X-band EPR spectra measured for [1·(L)4](ClO4)2 show rhombic type spectral features both in solid and solution phases, similar to that of the DHC2 isolated from G. sulfurreducens. Electrochemical data reveal good linear relationships for the first oxidation as well as FeIII/FeII redox couples with the pKa of axial ligand L. The FeIII/FeII redox couple in the diheme complex is shifted toward more positive than their monomeric analog which demonstrates easier reduction of diheme as compared to their monomeric analog. In order to substantiate the effect of interheme interaction in dihemes, a series of computational studies were performed. DFT studies reproduce the positive shifts of the FeIII/FeII redox couple on moving from monohemes to dihemes. It has also been observed that the redox potential could easily be tuned by changing the relative orientation of the two heme units in dihemes and also through the variation of iron-to-iron non bonding distance which eventually affects the interheme interactions. This could be well correlated with the variable redox properties shown by several DHC2 in nature. The atoms in molecules (AIM) analysis suggests long-range attractive dispersion forces between the heme units. The present investigation explores the effect of interheme interactions in diiron(III) and diiron(II) porphyrin dimers. The large variation in the structure and properties of dihemes, as compared to the analogous monohemes, accounts for the role played by interheme interactions in diheme.

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Figure 16. DAMQT 2.1.0 picture of [1·(Im)4]2+ highlighting critical points [(3,+3) CP in red and (3,+1) CP in green] between two heme units at fixed Fe···Fe separation of (A) 10.05 Å (as in most stable optimized geometry) and (B) 11.00 Å. Hydrogen atoms have been omitted for clarity.

EXPERIMENTAL SECTION

Materials. μ -Oxo- sy n -1 ,2- bi s[ 5 -(2 ,3, 7,8 ,12 ,13 ,1 7,1 8octaethylporphyrinato)iron(III)]ethane (1), 1,2-bis[(chloro){5(2,3,7,8,12,13,17,18-octaethylporphyrinato)}iron(III)]ethane (1· Cl2), 1,2-bis[(perchlorato){5-(2,3,7,8,12,13,17,18octaethylporphyrinato)}iron(III)]ethane (1·(ClO4)2), and 1,2-bis[(2,4,6-trinitrophenolato){5-(2,3,7,8,12,13,17,18octaethylporphyrinato)}iron(III)]ethane (1·(TNP)2) have been synthesized following the reported procedures.8c,d,10a Reagents and solvents were purchased from commercial sources and purified by standard procedures before use. Preparation of [1·(1-MeIm)4](ClO4)2. Complex [1·(L)4](ClO4)2, where X = Cl, ClO4, and TNP, was prepared using a general procedure; details are given for [1·(1-MeIm)4](ClO4)2 as a representative case. 1·ClO4 (60 mg, 0.043 mmol) was dissolved in 20 mL of distilled dichloromethane, and 1-methylimidazole (70 mg, 0.260 mmol) was added to it and stirred for nearly 5 min to form a deep red solution. The solution was then quickly filtered to remove any solid residue and was evaporated to dryness. The solid thus obtained was then dissolved in a minimum volume of benzene and carefully layered with n-hexane. After 5−6 days in air at room temperature, a dark crystalline solid was formed that was then collected by filtration, was washed well with mother liquor, and dried under vacuum. Yield: 58 mg (78%). UV−vis (dichloromethane) [λmax, nm (ε, M−1 cm−1)]: 415 nm (1.7 × 105), 545 (7.2 × 103). 1H NMR (CDCl3, 295 K): meso-H, −1.3, −8.6; CH3, −0.3, −0.2, 0.6, 2.1; CH2, 2.2, 3.5, 5.4, 6.1, 6.9, 7.2(2), 8.1; CH2(b),

suggest long-range attractive dispersion forces between the heme units which contribute to the through space heme-toheme interaction in diheme. Moreover, AIM analysis of uB3LYP/BS1 optimized geometry of [1·(Im)4]2+ at fixed Fe··· Fe separation of 11.0 Å was also done (Figure 16, trace B) which displays insignificant numbers of critical points between the two heme centers since they are substantially far from each other. Thus, such long-range dispersion forces between two heme centers in diheme are responsible for functional superiority of dihemes over monoheme analogs.

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CONCLUSION A large series of six-coordinated diiron(III) and diiron(II) ethane-bridged porphyrin dimers, as synthetic analogs of diheme cytochromes c, have been isolated and structurally characterized. Gradual addition of axial ligand L (L = imidazoles and pyridines) into pentacoordinated 1·X2 (X = ClO4,Cl and TNP) produces hexacoordinated diiron(III) porphyrin dimer [1·(L)4](X)2 which, in presence of excess I


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Inorganic Chemistry 16.8; −Ha, −7.0; −Hb, 15.9; −NMec, 13.5 ppm. EPR data: in dichloromethane (77 K), g1 = 1.53, g2 = 2.25, g3 = 2.86. Mössbauer: (α-Fe, 295 K): δ = 0.18 mm s−1, ΔEQ = 2.19 mm s−1. Preparation of [1·(Im)4](ClO4)2. Yield: 52 mg (72%). UV−vis (dichloromethane) [λmax, nm (ε, M−1 cm−1)]: 418 (1.6 × 105), 549 (9.9 × 103). 1H NMR (CDCl3, 295 K): meso-H, −1.9, −8.5; CH3, −0.9, −0.7, −0.1, 1.7; CH2, 2.9(2), 4.6, 5.3, 6.0, 6.2, 6.4 and 6.9; CH(b), 15.9; −Ha, −9.9; −Hb, 13.7; −Hc, 10.7 ppm. EPR data: in dichloromethane (77 K), g1 = 1.75, g2 = 2.27, g3 = 2.70. Mössbauer: (α-Fe, 295 K): δ = 0.19 mm s−1, ΔEQ = 2.12 mm s−1. Preparation of [1·(4-MeIm)4](ClO4)2. Yield: 51 mg (70%). UV−vis (dichloromethane) [λmax, nm (ε, M−1 cm−1)]: 408 (1.6 × 105), 539 (1.1 × 104). 1H NMR (CDCl3, 295 K): meso-H, −1.1, −10.6; CH3, −0.7, −0.6, 0.1, 1.7; CH2, 2.1, 2.2, 2.6, 4.4, 5.1, 5.7, 5.8, 5.9; CH2(b), 15.6; −Ha, −11.9; −Hb, 12.2; −Hc, 10.9; −4Meb, 12.9 ppm. EPR data: in dichloromethane (77 K), g1 = 1.75, g2 = 2.26, g3 = 2.67. Preparation of [1·(2-MeIm)4]Cl2. Yield: 44 mg (65%). UV−vis (dichloromethane) [λmax, nm (ε, M−1 cm−1)]: 411 (1.5 × 105), 556 (8.6 × 103). 1H NMR (CDCl3, 295 K): meso-H, −0.7, −4.6; CH3, 0.0, 0.2, 0.8, 1.7; CH2, 4.6, 5.4, 5.9, 6.4, 6.6, 6.8, 6.9, 7.8; CH2(b), 16.1; − Hb, 13.8; −Hc, 11.6 ppm. Mössbauer: (α-Fe, 295 K): δ = 0.16 mm s−1, ΔEQ = 1.55 mm s−1. Preparation of [1·(Py)4](TNP)2. Yield: 53 mg (80%); UV−vis (dichloromethane) [λmax, nm (ε, M−1 cm−1)]: 398 nm (1.7 × 105), 519 (1.4 × 104), 552 (1.3 × 104). 1H NMR (CDCl3, 295 K): meso-H, 0.8, −5.9; CH3, 1.2, 1.7, 2.1, 2.8; CH2, 6.1, 6.5, 6.7, 8.5, 9.7, 10.1, 10.4, 11.3; CH2(b), 18.3 ppm. Mössbauer: (α-Fe, 295 K): δ = 0.15 mm s−1, ΔEQ = 2.29 mm s−1. Preparation of [1·(4-CNPy)4](ClO4)2. Yield: 36 mg (65%); UV−vis (dichloromethane) [λmax, nm (ε, M−1 cm−1)]: 393 nm (1.6 × 105), 517 (1.4 × 104), 552 (1.1 × 104). Preparation of 2·(Py)4. Complex 2·(L)4 was prepared using a general procedure; details are given for 2·(Py)4 as a representative case. 1·ClO4 (50 mg, 0.036 mmol) was dissolved in 20 mL of distilled chloroform, pyridine (22 mg, 0.288 mmol) was added to it, and the mixture was stirred for nearly 10 min to form a deep red solution. The solution was then quickly filtered and was concentrated under reduced pressure. The solution was, then, carefully layered with acetonitrile. After 7−8 days in air at room temperature, dark crystalline solids were obtained which were collected by filtration, well washed with mother liquor, and dried under vacuum. Yield: 29 mg (54%); UV−vis (dichloromethane) [λmax, nm (ε, M−1 cm−1)]: 413 nm (2.0 × 105), 523 (1.4 × 104), 565 (1.3 × 104). Preparation of 2·(3-CNPy)4. Yield: 30 mg (51%); UV−vis (dichloromethane) [λmax, nm (ε, M−1 cm−1)]: 396 nm (1.9 × 105), 521 (1.4 × 104), 556 (1.2 × 104). Preparation of 2·(4-CNPy)4. Yield: 32 mg (55%); UV−vis (dichloromethane) [λmax, nm (ε, M−1 cm−1)]: 395 nm (1.8 × 105), 519 (1.5 × 104), 548 (1.3 × 104). Instrumentation. UV−vis spectra were recorded on a PerkinElmer UV/vis spectrometer. Electron paramagnetic resonance (EPR) spectra were obtained on a Bruker EMX EPR spectrometer. 1 H NMR spectra were recorded on a JEOL 500 MHz instrument. The spectra for paramagnetic molecules were recorded over a 100-kHz bandwidth with 64 K data points and a 5 ms 90° pulse. For a typical spectrum between 2000 and 3000 transients were accumulated with a 50-s delay time. The residual 1H resonances of the solvents were used as a secondary reference. 57Fe Mössbauer spectra were recorded using a Wissel 1200 spectrometer and a proportional counter. 57Co(Rh) in a constant acceleration mode was used as the radioactive source. Isomer shifts (δ) are given related to α-iron foil at room temperature. Cyclic voltammetric studies were performed on a BAS Epsilon electrochemical workstation in dichloromethane with 0.1 M tetra(nbutyl)ammonium hexafluorophosphate (TBAH) as supporting electrolyte and the reference electrode was Ag/AgCl and the auxiliary electrode was a Pt wire. The concentration of the compounds was in the order of 10−3 M. The ferrocene/ferrocenium couple occurs at E1/2 = +0.45 (65) V versus Ag/AgCl under the same experimental conditions.

X-ray Structure Solution and Refinement. Single-crystal X-ray data were collected at 100 K on a Bruker SMART APEX CCD diffractometer equipped with CRYO Industries low-temperature apparatus and intensity data were collected using graphitemonochromated Mo Kα radiation (λ= 0.71073 Å). The data integration and reduction were processed with SAINT software.25 An absorption correction was applied.26 The structure was solved by the direct method using SHELXS-97 and was refined on F2 by fullmatrix least-squares technique using the SHELXL-2014 program package.27 Non-hydrogen atoms were refined anisotropically. The hydrogen atoms were included in calculated positions. In the refinement, hydrogens were treated as riding atoms using SHELXL default parameters. CCDC 1824035−1824041 and 1837539 contain the supplementary crystallographic data for this paper. These data are provided free of charge by the Cambridge Crystallographic Data Centre. Computational Details. All DFT optimizations have been carried out by employing a B3LYP hybrid functional using the Gaussian 09, revision B.01, package software package.21 The method used was Becke’s three parameter hybrid exchange functional, the nonlocal correlation provided by the Lee, Yang, and Parr expression, and Vosko, Wilk, and Nuair 1980 correlation functional (III) for local correction.22 The basis set was LANL2DZ for iron atom and 6-31G** for carbon, nitrogen, oxygen, chlorine and hydrogen atoms, combination labeled as BS1. The coordinates are taken directly from the single crystal X-ray data and subsequently unconstrained geometry optimization of the molecules are done keeping the spinstate (low-spin) constant both for iron(III) and iron(II) porphyrin dimers. For 1D potential energy surface (PES) scan and for molecular orbital calculation, the coordinates are taken from the optimized geometry. Each one of the axial ligand from both the heme units was rotated in simultaneous steps of 10° keeping the corresponding other two axial ligands intact in their position. Solvent corrections were included in all the calculations using chloroform as solvent. Frequency calculations were also carried out on all optimized structures to ensure that there are zero or very few imaginary frequencies. Energies were taken from the Gaussian frequencies, and contain zero-point corrections at 298.15 K and 1 atm. The optimized geometry diagrams were made employing the Chemcraft software.28 Unless otherwise specified, the counteranions were removed for the full geometry optimization of [1·(L)4]2+ (where L is substituted imidazoles and pyridines) giving an overall +2 charge to the molecule with a overall spin multiplicity of triplet. For geometry optimization of 2·(L)4, the charge and spin multiplicity given was 0 and 1, respectively. To estimate the reduction potential for FeIII/FeII associated with 2Fe3+ + 2e− → 2Fe2+ occurring during diiron(III) porphyrin dimer reduction, the diiron(III) as well as corresponding diiron(II) i.e. 2e− reduced complex were geometrically optimized. The free energy change (ΔA0) associated with the 2e− reduction was calculated using the following equation: E0 = −ΔA0/nF, with F is the Faraday constant, and n is the number of electrons involved in the redox process FeIII/FeII which is 2 for dihemes and 1 for monohemes. The calculations provide absolute E0 values, which were subsequently reported as relative to SHE, by subtracting the IUPAC recommended 4.420 V value for the hydrogen semireaction.29 Similarly, the redox potentials for FeIII/FeII for monoheme complexes were calculated by geometry optimization of iron(III) and its 1e− reduced iron(II) porphyrin complex. For a better understanding of such heme−heme interaction linked reduction potential, a series of DFT calculations were done which involved variable iron−iron separation. The geometries of several conformers of [1·(Im)4]2+ were optimized with the iron to iron non bonding distance varying from 9.5 to11.0 Å in small steps. At 11.0 Å, both the diiron(III) as well as its diiron(II) analog, i.e., 2·(Im)4 was optimized and the potential for FeIII/FeII redox couple was calculated in a similar fashion described above. Similarly, the relative orientations of both the porphyrins rings were changed by varying the dihedral angle (θ’) between the heme units in [1·(Im)4]2+ from 0° (most stabilized geometry obtained through optimization) to 50° in either J


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Inorganic Chemistry directions. At 50°, the potential for FeIII/FeII redox couple for [1· (Im)4]2+ was similarly calculated.

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iron−sulfur, or copper redox centers. Chem. Rev. 2014, 114, 4366− 4469. (b) Paquete, C. M.; Louro, R. O. Unveiling the details of electron transfer in multicenter redox proteins. Acc. Chem. Res. 2014, 47, 56−65. (c) Bewley, K. D.; Ellis, K. E.; Firer-Sherwood, M. A.; Elliott, S. J. Multi-heme proteins: Nature’s electronic multi-purpose tool. Biochim. Biophys. Acta, Bioenerg. 2013, 1827, 938−948. (d) Mayfield, J. A.; Dehner, C. A.; DuBois, J. L. Recent advances in bacterial heme protein biochemistry. Curr. Opin. Chem. Biol. 2011, 15, 260−266. (e) Sharma, S.; Cavallaro, G.; Rosato, A. A systematic investigation of multiheme c-type cytochromes in prokaryotes. JBIC, J. Biol. Inorg. Chem. 2010, 15, 559−571. (f) Akutsu, H.; Takayama, Y. Functional roles of the heme architecture and its environment in tetraheme cytochrome c. Acc. Chem. Res. 2007, 40, 171−178. (g) Shi, L.; Squier, T. C.; Zachara, J. M.; Fredrickson, J. K. Respiration of metal (hydr)oxides by Shewanella and Geobacter: A key role for multihaem c-type cytochromes. Mol. Microbiol. 2007, 65, 12−20. (h) Stevens, J. M.; Daltrop, O.; Allen, J. W. A.; Ferguson, S. J. C-type cytochrome formation: chemical and biological enigmas. Acc. Chem. Res. 2004, 37, 999−1007. (2) Pereira, I. A.; Xavier, A. V. in Encyclopedia of Inorganic Chemistry; King, R. B., Ed.; John Wiley & Sons: New York, 2005, vol 5, pp 3360−3376. (3) (a) Watanabe, H. C.; Yamashita, Y.; Ishikita, H. Electron transfer pathways in a multiheme cytochrome MtrF. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 2916−2921. (b) Santos, T. C.; Silva, M. A.; Morgado, L.; Dantas, J. M.; Salgueiro, C. A. Diving into the redox properties of Geobacter sulf urreducens cytochromes: a model for extracellular electron transfer. Dalton Trans 2015, 44, 9335−9344. (c) Zhang, Y.; Liu, C.; Balaeff, A.; Skourtis, S. S.; Beratan, D. N. Biological charge transfer via flickering resonance. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 10049−10054. (d) Burggraf, F.; Koslowski, T. Charge transfer through a cytochrome multiheme chain: Theory and simulation. Biochim. Biophys. Acta, Bioenerg. 2014, 1837, 186−192. (e) Soares, C. M.; Baptista, A. M. Simulation of multihaem cytochromes. FEBS Lett. 2012, 586, 510−518. (4) (a) Breuer, M.; Rosso, K. M.; Blumberger, J. Electron flow in multiheme bacterial cytochromes is a balancing act between heme electronic interaction and redox potentials. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 611−616. (b) Fonseca, B. M.; Paquete, C. M.; Salgueiro, C. A.; Louro, R. O. The role of intramolecular interactions in the functional control of multiheme cytochromes c. FEBS Lett. 2012, 586, 504−509. (c) Paquete, C. M.; Louro, R. O. Molecular details of multielectron transfer: the case of multiheme cytochromes from metal respiring organisms. Dalton Trans 2010, 39, 4259−4266. (d) Zatsman, A. I.; Zhang, H.; Gunderson, W. A.; Cramer, W. A.; Hendrich, M. P. Heme-heme interactions in the cytochrome b6 f complex: EPR spectroscopy and correlation with structure. J. Am. Chem. Soc. 2006, 128, 14246−14247. (e) Mowat, C. G.; Chapman, S. K. Multi-heme cytochromesnew structures, new chemistry. Dalton Trans 2005, 34, 3381−3389. (5) (a) Pokkuluri, P. R.; Londer, Y. Y.; Duke, N. E. C.; Long, W. C.; Schiffer, M. Family of cytochrome c7-type proteins from Geobacter sulf urreducens: Structure of one cytochrome c7 at 1.45 Å Resolution. Biochemistry 2004, 43, 849−859. (b) Leys, D.; Meyer, T. E.; Tsapin, A. S.; Nealson, K. H.; Cusanovich, M. A.; Van Beeumen, J. J. Crystal structures at atomic resolution reveal the novel concept of “electronharvesting” as a role for the small tetraheme cytochrome c. J. Biol. Chem. 2002, 277, 35703−35711. (c) Matias, P. M.; Coelho, A. V.; Valente, F. M. A.; Plácido, D.; LeGall, J.; Xavier, A. V.; Pereira, I. A. C.; Carrondo, M. A. Sulfate respiration in Desulfovibrio vulgaris Hildenborough. J. Biol. Chem. 2002, 277, 47907−47916. (d) Einsle, O.; Stach, P.; Messerschmidt, A.; Simon, J.; Kröger, A.; Huber, R.; Kroneck, P. M. H. Cytochrome c nitrite reductase from Wolinella succinogenes. J. Biol. Chem. 2000, 275, 39608−39616. (e) Matias, P. M.; Coelho, R.; Pereira, I.; Coelho, A. V.; Thompson, A. W.; Sieker, L. C.; Gall, J. L.; Carrondo, M. A. The primary and three-dimensional structures of a nine-haem cytochrome c from Desulfovibrio desulf uricans ATCC 27774 reveal a new member of the Hmc family. Structure 1999, 7, 119−130.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.8b01368. X-ray structures, packing diagrams, and out-of-plane displacement plots of [1·(1-MeIm)4](ClO4)2, [1·(2MeIm)4]Cl2, [1·(4-CNPy)4](ClO4)2, and [1·(Py)4](TNP)2 (Figures S1−S8); X-ray structures, packing diagrams, and out-of-plane displacement plots of 2· (Py)4, 2·(3-CNPy)4 and 2·(4-CNPy)4 (Figures S9− S14); Mulliken spin density plots for [1·(Py)4]2+ and [1· (2-MeIm)4]Cl2 (Figures S15 and S16); UV−visible spectral change upon two-electron reduction of [1·(4MeIm)4](ClO4)2 (Figure S17); uB3LYP/BS1 optimized structures of [1·(2-MeIm)4]Cl2, 2·(2-MeIm)4, [1· (Py)4]2+, and 2·(Py)4 (Figure S18−S22), Cartesians of all optimized geometries; selected bond distances (Å) and bond angles (deg) for the complexes along with crystal data and data collection parameters (Tables S1− S5); relative energies of axial ligand orientations (Table S6); and selected structural and geometrical parameters of optimized species with relative energies (Tables S7− S12) (DOCX) Accession Codes

CCDC 1824035−1824041 and 1837539 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/ cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Authors

*(S.P.R.) E-mail: [email protected]. Telephone: (+91)-512259-7251. Fax: (+91)-512-259-7436. *(D.K.) E-mail: [email protected]. ORCID

Sankar Prasad Rath: 0000-0002-4129-5074 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Science and Engineering Research Board (SERB), New Delhi, and Council of Scientific and Industrial Research (CSIR) New Delhi for financial support. F.S.T.K. thanks University Grants Commission (UGC), India, and S.B. thanks Innovation in Science Pursuit for Inspired Research (INSPIRE) of Department of Science and Technology, New Delhi for their fellowships.

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DEDICATION Dedicated to Professor Akhil R. Chakravarty on the occassion of his 65th birthday. REFERENCES

(1) (a) Liu, J.; Chakraborty, S.; Hosseinzadeh, P.; Yu, Y.; Tian, S.; Petrik, I.; Bhagi, A.; Lu, Y. Metalloproteins containing cytochrome, K


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Inorganic Chemistry

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