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EPR Characterization of the Triheme Cytochrome from Geobacter sulfurreducens Nina Ponomarenko, Jens Niklas, Phani Raj Pokkuluri, Oleg G. Poluektov, and David Michael Tiede Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00917 • Publication Date (Web): 03 Jan 2018 Downloaded from http://pubs.acs.org on January 3, 2018

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Biochemistry

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Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

EPR Characterization of the Triheme Cytochrome from Geobacter sulfurreducens Nina Ponomarenko*,1, Jens Niklas1, P. Raj Pokkuluri2, Oleg Poluektov1, David M. Tiede*,1 1

Chemical Sciences and Engineering Division and 2Biosciences Division Argonne National Laboratory 9700 South Cass Ave. Argonne, IL 60439

*Corresponding Authors: [email protected], [email protected]

ABSTRACT Periplasmic cytochrome A (PpcA) is a representative of a broad class of multi-heme cytochromes functioning as protein “nanowires” for storage and extracellular transfer of multiple electrons in the δ-proteobacterium Geobacter sulfurreducens. PpcA contains three bis-His coordinated hemes held in a spatial arrangement that is highly conserved among the multi-heme cytochromes c3 and c7 families, carries low potential hemes and is notable for having one of the lowest number of amino acids utilized to maintain a characteristic protein fold and site specific heme function. Low temperature X-band EPR spectroscopy has been used to characterize the electronic configuration of the Fe(III) and the ligation mode for each heme. The three sets of EPR signals are assigned to individual hemes in the 3D crystal structure. The relative energy levels of the Fe(III) 3d orbitals for individual hemes was estimated from the principal g values. The observed g tensor anisotropy was used as a probe of electronic structure of each heme and differences were determined by specifics of axial ligation. To ensure unambiguous assignment of highly anisotropic low spin (HALS) signal to individual hemes, EPR analyses of iron atom electronic configurations have been supplemented with investigation of porphyrin macrocycles by 1D 1H NMR chemical shift patterns for the methyl substituents. Within optimized geometry of hemes in PpcA the magnetic interactions between hemes were found to be minimal, similar to c3 family of tetraheme cytochromes.

2


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Biochemistry

Key words

Periplasmic cytochrome A, PpcA, EPR, 1H NMR, triheme cytochrome, heme coordination, Geobacter sulfurreducens

INTRODUCTION Periplasmic cytochrome A (PpcA) from the metal-reducing δ-proteobacterium Geobacter sulfurreducens is a small, 9.57 kDa, triheme cytochrome that belongs to a family of periplasmic multiheme, c-type cytochromes which function in energy-conserving electron transport, connecting microbial metabolism to extracellular redox chemistry 1-3. Multi-heme cytochromes are of special interest because of their function as protein “nanowires” for storage and extracellular transfer of multiple electrons in both metal dissimilatory

4-6

and photosynthetic 7, 8

microorganisms, and for their potential use as the building blocks in designed architectures for coupling single electron transfer to multi-electron catalysis 9-11. The heme prosthetic group in c-type cytochromes are covalently attached to the polypeptide matrix through thioether linkages between heme vinyl side chains and protein cysteine residues in a conserved amino acid binding motif CXXCH

12, 13

. The histidine residue in the heme-

binding pattern functions as the first axial ligand (fifth ligand overall) to the central iron atom of heme, whereas another histidine, or different amino acid, located elsewhere in protein chain, forms the second axial ligand (sixth ligand overall). The coupling of hemes in an amino acid milieu creates the proper spatial arrangement required for long range electron transfer with sequential changes of the metal oxidation state 14, 15. The covalent binding confers stability to the cytochrome structure, fixes the positions of hemes, and minimizes the number of amino acid residues required to define the heme binding pocket16. As a consequence, c-type cytochromes gain the ability to organize multiple heme cofactors on a short extent of a polypeptide chain. Most of the multiheme cytochromes have a tight globular folding with two characteristic diheme structural modules: perpendicular (or T-shaped) arrangement and parallel stacking of the porphyrin planes

17, 18

. Since the efficiency of electron transfer is highly dependent on the

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distance between metal centers, the close packing of heme cofactors in multiheme cytochromes ensures the orbital overlap and efficient interaction between metal cores 19, 20. A prominent feature of PpcA is the dense packing of hemes within the protein matrix, Figure 1 21, 22

. Consisting of only 71 amino acids, this protein is notable for using a relatively short peptide

chain to bind 3 heme cofactors and maintain a well-defined structural motif. Crystallographic structures show that overall arrangement and orientation of the hemes in PpcA, as well as other representatives of triheme cytochromes c7, are homologous to the broader and initially characterized tetraheme cytochromes c3 with the only exclusion being heme II along with the corresponding polypeptide chain segment 22-25. Owing to these similarities the nomenclature for hemes in PpcA retains the same as in the c3 cytochromes, with numbers following the order of attachment to the CXXCH motif in the polypeptide chain, Figure 1.

Figure 1. Ribbon representation of the PpcA cytochrome from G. sulfurreducens. The ribbon is colored in the range from blue (N terminus) to red (C terminus). The three heme groups are designated by Roman numerals according nomenclature common with tetraheme c3 cytochromes 4


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Biochemistry

to account for the order of attachment to the polypeptide chain. The inset on the left shows the heme diagram with the IUPAC nomenclature for identifying priciple axes system and geometric parameters determining orientation angle φ and interligand angle β used in this study. When looking at the heme from the sixth coordination site, the heme substituents are arranged in a clockwise order with increasing numbers corresponding to β-positions of the pyrrole rings. In particular, methyl groups are substituents at 2, 7, 12, 18 positions. Carbons with the implied numbers 5, 10, 15 and 20 located at generic meso-positions. The X axis goes through nitrogen atoms N2 and N4, Y axis through N1 and N3 of corresponding pyrrole rings. The Z axis is aligned along the normal to the heme plane. The orientation angle φ is measured from the X axis in counterclockwise direction to and past the Y axis until the average between the two histidine ligands positions. The interligand angle β is a dihedral angle between imidazole planes of histidine ligands. Functionally, PpcA is characterized by the relatively low redox potentials for each of the three heme cofactors. The heme midpoint potentials are found to lie within a rather narrow range, from the lowest of heme I equal to -162 mV to the highest potential of heme IV, around -133 mV 26. Nuclear magnetic resonance (NMR) chemical shift perturbation measurements have demonstrated the presence of specific binding sites on the PpcA surface for small molecule redox substrates

27, 28

and partner redox proteins

29

. Similarly, PpcA dimerization induced by anionic

porphyrin binding also points toward an electrostatic surface that is mapped for recognition at a single surface location

30

. Combined, these results suggest the design for directional electron

transfer controlled through PpcA. From this perspective, the mechanisms for protein tuning of heme cofactors for redox and direct electron transfer function are of significant interest. Evidence for site-selective tuning of the heme cofactors in PpcA is seen by the heme methyl group proton NMR chemical shift patterns that are distinct, and readily distinguishable for each of the three heme cofactors

27, 28

. The differing chemical shift patterns observed for the heme

cofactors in both the cytochromes c3 and c7 families are significant since they provide a measure of the modulation of electron spin densities at atomic sites on the porphyrin ring which are found to be characteristically tuned for each heme cofactor 31-35. A direct approach for characterization of site-specific electronic structure configuration of ferric hemes is through electron paramagnetic resonance, EPR, spectroscopy 36-38. EPR spectroscopy is frequently applied to gain insight into processes governing electron transfer in paramagnetic multiheme centers, in particular to reveal electronic composition and redox interactions of prosthetic groups in tetraheme cytochromes 39-43. This technique is also beneficial for analysis of the ligand coordination and electronic structures of the heme cofactors of triheme PpcA. At ambient conditions all of the PpcA hemes are oxidized, exhibiting Fe(III), and are therefore in

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an EPR-detectable paramagnetic state. EPR spectroscopy provides the means for examination the electronic configuration of the ferric heme focusing on the immediate vicinity of the iron and distribution of electron density determining it (or the orbital hole accompanying it) without interference from a diamagnetic protein milieu 44, 45. EPR spectroscopy is a useful companion to NMR chemical shift data, providing a direct measure of magnetic properties of the heme iron. With these advantages EPR analyses can complement the existing investigation of magnetic properties of PpcA cytochrome undertaken by NMR35,

46

and characterize the modulation of

heme iron electronic structure by protein site coordination geometries with higher precision. The use of protein atomic coordinate-based spectroscopic modeling and analysis has been shown to convey a fuller understanding of local protein site tuning of metal cofactor electronic structures.47 In particular, insights into cofactor electronic configurations have been achieved by using EPR analysis in combination with information about molecular structure

48, 49

. The

availability of crystal and solution structures of PpcA 21, 22, 35 gives the opportunity. Thus, in this study we have utilized X-band EPR spectroscopy in combination with 1D 1H NMR analysis of chemical shifts of heme methyl groups to characterize electronic structure of triheme cytochrome PpcA and define the relationship between electronic configuration and coordination of individual hemes. The results provide a comprehensive examination of the site-specific tuning of PpcA heme cofactors.

MATERIALS AND METHODS Expression and purification of cytochrome. PpcA have been produced by recombinant expression in host organism Escherichia coli BL21. The heterologous expression system included template plasmid pVA203 and accessory plasmid pEC86 bearing cytochrome c maturation genes

50

. The presence of this plasmid ensured the heme synthesis with subsequent

transport through the cellular membrane and proper assembly of cytochromes in the periplasm. The Stratagene QuickChange II mutagenesis kit was used for introducing mutations into template pVA203. Purification of recombinant PpcA cytochromes was performed as described in 51

. Size-exclusion chromatography using Superdex 75 column (GE Healthcare) equilibrated in 10

mM Tris-HCl buffer (pH=7.5), 100 mM NaCl was added as a final step in purification.

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Biochemistry

EPR Experiments. For the EPR measurements, ≈ 1 mM ferric PpcA solutions were prepared in a Tris-HCl buffer (pH 7.5) containing 100 mM NaCl. The solutions were filled into 4 mm o.d. quartz tubes, which were sealed under nitrogen atmosphere to maintain anaerobic conditions for the EPR measurements. Continuous wave (CW) X-band (9 - 10 GHz) EPR experiments were carried out with a Bruker ELEXSYS II E500 EPR spectrometer (Bruker Biospin, Rheinstetten, Germany), equipped with a TE102 rectangular EPR resonator (Bruker ER 4102ST). A helium gas-flow cryostat (ICE Oxford, UK) and an ITC503 from Oxford Instruments, UK, were used for measurements at cryogenic temperatures. Data processing was done using Xepr (Bruker BioSpin, Rheinstetten) and Matlab 7.11.2 (The MathWorks, Inc., Natick, Massachusetts, USA) software. The EPR spectra were corrected by subtraction signals of the cavity background and the EPR tube containing buffer recorded under identical conditions. Simulations were performed using WINEPR SimFonia software provided by Bruker and EasySpin software package (version 5.1.12) 52. Correlation between EPR g-tensor values and ligand-field parameters. The ligand-field correlation analysis, based on the formalism introduced by Griffith 53 and developed by Taylor 54 has been used to find the rhombic (V/λ) and axial (∆/λ) crystal field parameters. The corresponding V/∆ ratio was applied for determination of system rhombicity and analysis of hemes coordination. Detailed description of the model for the heme molecular orbitals characterization and correlation of empirical g-value to the axial ligand geometry can be found in Supporting Information. The freeware programs developed at University of Arizona were utilized

55

for calculation of ligand field parameters based on experimentally defined g values

and verification of the optimum combination of g values satisfying normalization conditions required by the above formalism (gz2+ gy2+ gx2 = 16). NMR Analysis. For NMR studies PpcA cytochrome was transferred to a sodium phosphate buffer (pH 7.5), 100 mM NaCl prepared in 99.9% deuterium oxide. Sequential dilution and diafiltration with Amicon ultra centrifugal filter units were used to replace the buffer and concentrate the sample to ≈ 1 mM. The 1D 1H NMR spectra were acquired at 25°C in a Bruker Avance III 500 MHz spectrometer by collecting 128 K data points with 256 scans and a sweep width of 20 kHz. The spectra were processed using software TOPSPIN (Bruker Biospin, Karlsruhe, Germany) and calibrated using the water signal as internal reference. The chemical

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shifts are reported in parts per million (ppm), relative to tetramethylsilane. The resonances of PpcA have been assigned previously through the examination of 1D 1H,

13

C-HSQC NMR and

2D 1H-NOESY, 2D 1H-TOCSY data 21, 35. The model representing the paramagnetic hyperfine shifts as a function of axial ligand nodal plane orientation was applied for analysis of the 1H NMR spectrum of the PpcA molecule

56, 57

.

In this model the Hückel calculations of the electron densities at the β-pyrrole and mesopositions of the heme were used to relate the contact shifts for each methyl group protons to axial ligands positions

56

. The concept of counter-rotation of the in-plane g-tensor axes and planar

axial ligands away from the N-Fe-N axes of the heme

58

was applied for estimation of the

pseudocontact shifts. The ligand plane orientation angle φ was measured from the X axis (going through N2-Fe-N4 of porphyrin plane) toward and past the Y axis (N1-Fe-N3), while the Z axis was aligned along the normal to the heme plane. The dihedral angle β defined the mutual orientation of imidazole planes of histidine ligands (Figure 1). The graphical representation of this model, relating the order and relative spacings of the heme resonances to averaged angle between the two imidazole planes of histidine ligands 56, can be acquired using the program Shift Patterns 57.

RESULTS AND DISCUSSION EPR analysis. The CW X-band EPR spectra of the PpcA cytochrome recorded at 10 K represent the resonance signals typical for low spin (S=1/2) ferric heme complexes, Figure 2. In agreement with previous EPR analysis of hemes with different symmetry distortions

37, 38

the observed

spectrum consists of two types of signals. The first is associated with signals around gmax ≈ 3.50 and is known as highly axial low spin (HALS), while the second type corresponds to the “conventional” rhombic low-spin heme complex with gmax ≈ 2.96. The significant structural aspect of low spin heme complexes is coordination by two axial ligands, in particular by two histidines (bis-His coordinated). For this type of heme ligation the interrelation between the structural factors and heme electronic properties is well defined on metalloproteins and synthetic model porphyrins 37, 38. According to those investigations the relative orientation of the imidazole planes of heme coordinating histidine residues determines the type of EPR signal. Hemes with the close to perpendicular alignment of imidazole planes generate HALS or Type I EPR

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Biochemistry

spectrum with large gmax value (gmax > 3.2) and increased g anisotropy. Hemes with close to parallel orientation of two coordinating imidazole planes (dihedral angle is less than 70°) produce typical rhombic or type II EPR spectrum with lower gmax value, the small g anisotropy and readily observable all three principal values of a g-tensor. The electronic ground-state configuration is (dxy)2(dxz,dyz)3 for which gz>gy>gx with gz oriented near the normal to the heme plane (depicted in Figure S1 of SI)

38, 59

. The above concepts along with the experimentally

observed g values and structural parameters of PpcA molecule

21, 22, 35

, namely, dihedral angles

between imidazole planes of coordinating histidines for each heme, Table 1, were used for analysis of the EPR spectra and assignment of the signals to the individual hemes I, III and IV. There are some variations in dihedral (β) and orientation (φ) angles between published structures of PpcA, depending upon the methods and sample states used for structural determination. The orientations of the heme axial ligands are seen to differ for ferric PpcA when determined by Xray crystallography compared to NMR solution structures, presumably due to the presences of bound deoxycholate molecule that required for obtaining cyrstals21, 22. At the same time, in NMR solution structures the geometry of axial ligands is significantly influenced by the redox state of heme iron, as the key redox-linked conformational changes in ferric relative to ferrous PpcA are detected in orientation of the average histidine plane and in the interligand angle between the imidazole rings

21, 35

. Accordingly, in the following, the compiled structural information is

considered with respect to the resolved EPR properties of PpcA heme cofactors.

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2.96

5 mW 80 mW

3.24 3.50

x 15 2.21 2.63 1.47 2.03

200

300

400

500

600

Magnetic field, mT Figure 2. CW X-band EPR spectra of triheme cytochrome PpcA recorded at temperature10 K, microwave powers of 5 mW and 80 mW. g-values are shown for the individual peaks. Insert: additional line at high field regions is visible with a microwave power of 80 mW.

Table 1. Angles between imidazole ring planes of the axial ligands for each heme in PpcA Heme number

Ligands

Dihedral angle between imidazole planes Crystal structure, ferric 22

Solution structure, ferrous 21

β angle by 13 C NMR, ferric 46

β angle by 1 H NMR, ferric 35

Solution structure, ferric 35

Heme I

His17 His31

57°

50°

74°

70°

56.7° (13.4)

Heme III

His20 His55

22°

23°

42°

25°

73.9° (11.3)

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Biochemistry

Heme IV

His47 His69

89°

80°

84°

81°

58.8° (11.5)

Directly from the experimental EPR spectrum all three principal values of the g-tensor can be obtained only for one heme. The spectrum with the g-values gx=1.47, gy=2.21, gz=2.96, depicted in Figure 2, has relatively small g-tensor anisotropy (gz-gx) and belongs to the group of hemes with rhombic spectrum, characteristic for parallel or near-parallel alignment of the axial ligands 37

. Temperature dependence analysis shows that the EPR signals with the corresponding

positions and relative intensities were detectable up to 70 K, Figure S2, while the adjacent peaks vanish with increasing the temperature higher than 30 K. This observation confirms the correspondence of this set of principal g values to a single heme with a type II ferric heme center. Since the smallest angle between two imidazole planes is seen for the bis-His ligands for heme III, His 20 and His 55, we can explicitly assign heme III as having structural parameters that best correspond to the observed rhombic EPR spectrum (Table 1). The NMR-determined inter-ligand angle for ferrous heme III of 23o in the solution structure 21 is only slightly different from the 42o obtained in calculations based on the energy splitting of the ferric PpcA molecular orbitals 46. Thus, we can assign the rhombic type of EPR spectrum, to heme III with a high degree of confidence. The broad low field peaks with gz=3.50 and gz=3.24 detected at low magnetic field are characteristic for the HALS signals. Our assignment of these peaks is based on the structural parameters of the remaining two hemes, listed in Table 1. Note that the low intensity of these “large gmax” signals when compared to rhombic ones is caused by their higher g-tensor anisotropy (gz-gx). For this reason HALS signals are usually relatively weak and only the largest principal g value is readily detectable, while signals at gy and gx values are often broadened by the g-strain and they are barely visible or missing in EPR spectra 60-62. In addition, even gz band of HALS hemes can only be observed at very low temperatures (4–20 K) and these types of signals tend to have a higher microwave power saturation profile compared to the rhombic resonances due to more efficient spin-lattice relaxation (shorter T1e) of the iron center

62, 63

.

Therefore, the nature of HALS signals prevents direct assignment of all three g values. EPR measurements at higher microwave power, 80 mW, found that, while the signal saturation

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became apparent for the rhombic heme III, the low magnetic field peaks of HALS hemes intensified and displayed better resolved details, Figure 2. In addition, at high microwave power a broad signal was observed around g=1.15-1.20, as illustrated by the inset in Figure 2. We tentatively assign this signal to the HALS species of Heme IV. The signal is relatively broad and weak, and hence the possibility exists that at least some part of this signal is due to background signal(s) not associated with the protein. However, the g-value, the low amplitude and broad line shape are in good agreement with previous reported signals of HALS signals of ferric iron 38, and with well-established normalization requirements for the principal g-values of HALS species (see below). Thus, despite the saturation of EPR signals for rhombic heme III, this power was more favorable for observing the remaining HALS signals and was chosen as a compromise allowing concurrent analysis of all three hemes (Figure 3).

2.96

3.24 3.50 1750

2000

2250

1.22

2.21 1.47 2.03

2000

2500

3000

4000

5000

PpcA Heme I Heme III Heme IV Simulation 6000

Magnetic field, G

Figure 3. CW X-band EPR spectrum of the low-spin triheme cytochrome PpcA recorded at 10 K with power 80 mW and the simulated spectrum (sum of simulation of hemes I, III, IV) offset slightly for clarity. g-values are shown for the individual peaks. Insert represent low field region with individual hemes contributions to simulated spectrum. 12


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Biochemistry

The assignments of the HALS signals to particular hemes were also based on the mutual orientation of axial ligands from structure models

21, 22, 35

. The highest gz value of 3.50

corresponds to heme IV, since the dihedral angle between imidazole planes of this heme is the largest. It equals to 80° in the NMR solution structure of ferrous diamagnetic PpcA

21

or even

89° in an X-ray crystal structure for ferric oxidation state 22. Despite the fact that calculation of this angle from ferric PpcA NMR data produced a considerably lower value, around 58.8°, it remained higher than that calculated for heme I (56.7°) 35. The signal at gz=3.24, located close to the spectral region between HALS and regular rhombic low spin heme types of EPR spectra, was assigned to the remaining heme I. As determined in solution structure of ferrous PpcA, the angle between its axial ligands His17 and His31, has a value which lies between those of the other two hemes, 50°, making it a probable candidate for having the rhombic type of EPR spectrum. Further, from analysis of the 13C NMR paramagnetic shifts of the heme substituents for the ferric PpcA, the dihedral angle of heme I His ligands was concluded to be 74.0 degrees

35, 46

, which would be a clear indication of a HALS type of EPR

signal. Simultaneously, in the crystal structure for ferric oxidation state the angle between imidazole planes of His ligands of heme I equals 57°. This is exactly the interligand angle at which the switch between different EPR signal types occurs, according to studies of model porphyrins 64. NMR analysis. To have more conclusive estimation of magnetic anisotropy levels for individual hemes we supplemented the analysis of Fe(III) electronic configuration with investigation of the surrounding porphyrin macrocycle by 1H NMR. This combined approach allows characterization of the magnetic anisotropy within the entire heme, since both, the electronic parameters of unpaired electron on iron, gmax and ligand-field anisotropy V/∆, and the NMR paramagnetic shifts δ, experienced by the porphyrin nitrogen atoms, peripheral carbon and methyl groups, are governed by the distribution of electron spin density residing on the heme core 58, 65-68. Two types of chemical shifts, the isotropic Fermi contact shifts, originating from direct delocalization of the unpaired spin onto the iron-bound ligand, and dipolar or pseudocontact shifts, associated with the through-space interactions and orientation of the magnetic axes, contribute to a heme 1H NMR

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spectrum. While both types are influenced by the orientation of the nodal plane of the axial ligand, the contact shifts have significant domination in the allocation of four methyl group signals induced by an external magnetic field 56, 69. The downfield region of the 1H NMR spectra of PpcA in the air-oxidized state (Fe(III)) is depicted in Figure 4, with key results summarized in Table 2 (methyl groups are numbered according to the IUPAC-IUB nomenclature for heme substituents

70

, see also Figure 1). The

arrangement of methyl group shifts for heme III is 7CH3>12CH3>2CH3>18CH3, different from the two other hemes, Figure 5. According to the Shift Patterns program, which calculates the correlation between contact chemical shift and axial ligands orientation angle (measured from the X axis going through N2-Fe-N4 of the porphyrin plane)

57

, the observed order of methyl

group chemical shifts corresponds to angle in the range of 136 - 158°, Figure S3. This interval includes the orientation angle, around 143˚, known from crystal structure of PpcA, Figure 5. When in addition to the pattern of methyl group shifts the relative spacing between them is taken into account, the orientation angle approaches 154° (Figure S3), which is close to the averaged position of ligand planes in solution structure of reduced PpcA

21

. Notably, for heme III the

dependence of observed and calculated shifts from position of axial ligands best matched the systems having two ligands in parallel planes.

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Biochemistry

Figure 4. 1H NMR spectrum of the low-spin triheme cytochrome PpcA at pH=7.5, 100 mM NaCl, 25º C, 500 MHz and the assignment of the paramagnetic shifts of the four heme methyl groups at positions 2, 7, 12, and 18. Table 2. 1H chemical shifts of heme methyl groups in cytochrome PpcA at pH=7.5, 100 mM NaCl Chemical shift, ppm Group

Heme I

Heme III

Heme IV

21CH3

17.64

11.94

14.75

71CH3

10.14

17.93

10.44

121CH3

20.96

12.00

19.26

181CH3

15.53

0.65a

14.55

Mean(Deviation)

16.07(4.54)

10.88(7.30)

14.75(3.60)

Span of CH3 shifts

10.81

17.28

8.82

a

Value taken from Morgado et al., 201735 .

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For Heme I and Heme IV the methyl group shifts display the identical pattern 12CH3>2CH3>18CH3>7CH3, Figures 5 and 6, which originates from the comparable spin density distribution in these two hemes and implies the similar types of the heme axial coordination. This observation is in line with the assignments of both hemes to the same type of ferric heme center based on the analysis of EPR spectrum discussed above. While the order of methyl resonance shifts for two hemes is the same, the relative spacing is different, Figure 5, and Table 2. For heme IV the spread of the methyl groups’ shifts of 8.81 ppm is smaller than the 10.81 ppm spread of shifts measured for heme I. The revealed difference in spacing of paramagnetic shifts and the broader interval for heme I is a proof of more anisotropic electron spin density distribution in its porphyrin ring relative to heme IV. As known from the study of the low-spin Fe(III) model hemes and heme proteins, the distribution of the contact shifts for methyl groups depends on the interligand angle. While the mixing of the two heme π orbitals increases together with interligand angle, the energy difference between them, ∆Eπ, becomes smaller, which in turn decreases the spread of methyl resonances of the heme 71. Thus, the more compact heme methyl shift pattern indicates more uniform distribution of unpaired electron spin density at the four pyrrole groups, consistent with stronger axial coordination of Fe(III) 72-74.

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Biochemistry

Figure 5. 1H NMR chemical shift patterns observed in PpcA with corresponding front views of individual hemes. The 5th ligand, histidine from CXXCH binding motif, is lying behind the porphyrin planes. Orientation of histidine ligands represented according to crystal structure 22. The ligand orientation angles are measured from the X axis in counterclockwise direction to and past the Y axis until the average between the two histidine ligands position. Consequently, for all three hemes the order and relative spacing of methyl chemical shifts, reflecting anisotropy of heme measured by NMR, are in good agreement with the EPR analysis. At the same time, there is no direct correlation between the gz value of individual hemes, or their ligand-field derived anisotropy, with the averaged heme methyl chemical shift δ. Heme I, characterized by the intermediate values of gz and rhombicity index V/∆, has slightly larger average shift than heme IV (16.07 and 14.75 ppm correspondently, as listed in Table 2). A potential reason for deviation from linear correlation of chemical shift and heme anisotropy is the distortion of the porphyrin plane. As was shown in previous studies of nitrophorins, cytochrome c-552 and c-551 mutations, increasing of heme ruffling resulted in closer contact of molecular orbitals and the rise in the energy of the dxy orbital, which in turn caused the decrease in axial ligand-field term ∆ and higher rhombicity

63, 75

. Within the PpcA molecule heme IV is

the most puckered one according computation implemented by Shelnutt’s normal-coordinate structural decomposition analysis in

24

. Concomitantly, ruffling, the most significant type of

porphyrin puckering, was found comparable for heme I and IV, even a little higher for heme I, therefore it cannot be the main cause for the lack of correlation. One of the reasons for the observed deviation is the nonlinear functional dependence of the angle between two axial ligands and paramagnetic shifts of methyl groups. The detected, as well as predicted, spread of the methyl resonances is maximal when two axial ligand planes are parallel, but decreases fairly rapidly with increasing dihedral angle between them, especially when it approaches 90°

56

. At the same time dipolar or through-space interactions, reflected in the

pseudocontact shifts, remain nearly the same, or become more significant due to the large anisotropy of the g-tensor of the ferric hemes with this type of axial ligands alignment

56

. This

counteraction can be intensified by the structural distinctions of hemes, particularly in coordination of heme I. While the imidazole rings of five His ligands have close to perpendicular orientation relative to porphyrin planes, the imidazole of His17, the 6th ligand to heme I, is tilted to 62°

22

. This could lead to off-axis ligation and change in amplitude of methyl resonances

shifts. In addition, according to calculations for elucidation of the effect of axial ligands

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Page 18 of 30

orientation on paramagnetic shifts in 56, the sequence pattern and distribution range of chemical shifts for heme I would require a different orientation angle, in intervals 0º-3º or 135º-180º (when the interligand angle is around 70º), than determined from the averaged angle between axial ligands in solution (68º or 83º in oxidized and reduced PpcA, respectively) and crystal (110º) structures. This observation leads to assumption that axial ligands do not equally contribute in the coordination of iron. The orientation of His 17, positioned at 146º from X axis (Figure 5) in crystal structure, is within the required interval, indicating the domination of this ligand in coordination of heme I. Some flexibility of His 17 within PpcA molecule also can have an influence. While other coordinating histidines have at least one hydrogen bond to an amino acid in the polypeptide chain, His 17 is hydrogen-bonded to a water molecule and that is the longest hydrogen bond formed by His residue, equal to 2.92 Å

22

. The polypeptide segment

surrounding heme I and including His17 is the most dynamic part of PpcA in the ferrous and ferric oxidation state

21, 35

. The main difference between two solution structures of the PpcA as

well found in orientation of the ligand to heme I

35

. Certainly all these peculiarities of heme I

coordination can affect the magnitude of its methyl group chemical shifts and cause deviation from linear correlation with ligand field parameters. Electronic parameters and geometric structure. Further adjustment in assignments of g tensor principal values were based on the complete EPR spectrum simulations. The best fit of the experimental spectrum is shown in Figure 3. For simulation, the gy and gx parameters of HALS signals were estimated based on the normalization requirement gz2+gy2+gx2=16

54, 61

and

comparison with the previously reported bis-His ligated model systems or corresponding heme proteins having similar coordination and gz values

36, 37, 63, 76

. The complete EPR envelope of

PpcA was simulated with derivative Gaussian line shapes as a sum of spectra from three independent hemes (Figure 3, 7) with principal g values given in Table 3.

Table 3. The g-tensor principal values and ligand-field parameters determined by simulation of the X-band EPR spectra of individual hemes from PpcA cytochrome

Heme I

gz

gy

gx

V/λ

∆/λ

∑(a,b,c)2

V/∆

3.24

1.98

1.22

1.21

3.89

0.997

0.31

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Biochemistry

Heme III

2.96

2.21

1.47

1.77

3.40

0.997

0.52

Heme IV

3.50

1.54

1.15

0.88

8.76

0.999

0.10

The magnetic properties of the PpcA cytochrome have been interpreted using the formalism of Taylor developed from the t2g hole theory of Griffith

53, 54

, which relates the principal g values

and the ligand-field parameters of the Fe(III) heme center. The rhombic splitting parameter V/λ and tetragonal splitting parameter ∆/λ for each heme, defined from the experimentally determined and calculated g values, are listed in Table 3. The deduced relative energies of the iron 3d orbitals are illustrated in Figure S4 (Supporting Information). For the three g-tensor values observed for heme III, gz =2.96, gy =2.21, gx =1.47 (Table 3), the corresponding ligand-field parameters are V =1.77λ and ∆ = 3.4λ, respectively (with λ= 279 cm-1 69

). These parameters are in agreement with symmetry expected from the geometry of axial

ligands in quasi-parallel orientation and a strong rhombic field influencing the Fe3+ of the heme group. The g-tensor anisotropy (gz - gx) is smaller than for two other hemes, but doesn’t reach the potential minimum for hemes. The numerical value of rhombic distortion V/λ signifies the slight deviation from co-planar alignment of axial ligands, as V reaches 2λ when imidazole planes of histidine ligands are parallel to each other 77. In addition, for the low-spin ferric porphyrins with bis-imidazole ligands having small dihedral angle the calculated from the ligand field parameters rhombicity index V/∆ denotes orientation of imidazole planes relative to Fe-N bonds

37, 78

. The

systematic decrease in this index was observed as histidine ligands were rotated away from eclipsing (φ = 0˚) to bisecting a Fe-N vector configuration (φ = 45˚) 48, 79. According to the model developed in 78, 79 for prototypical porphyrin derivatives, the numerical value V/∆ = 0.52 received for heme III, would correspond to 32˚ - 36˚ deviation of ligand position from the closest Fe-N vector. The complementary angle equals to 146˚ and coincides with the middle of interval 135˚ 158˚ (angle measured from N2-Fe-N4 axis in counterclockwise direction) in orientation angle determined by 1H NMR and discussed before. For the heme I only gz=3.24 was unambiguously detected experimentally. An estimate of gx was extracted from the spectrum taken at power 80 mW, though the component was rather broad and both hemes, I and IV, possessing large g tensor anisotropy, could have gx in this region. Thus, the two other g tensor values were determined by the EPR simulation as well as taking into

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account the previously mentioned normalization condition, as gy=1.98, gx=1.22, Table 2. The derived ligand field parameters were V = 1.21λ, ∆ = 3.89λ, which gives V/∆ = 0.32, demonstrating that this heme has a weak axial system. The determined heme IV parameters V = 0.88λ and ∆ = 8.76λ are indications of the significant spectral anisotropy. The low V/∆ index, equal to 0.10, reflects the almost degenerate energy levels of the dxz and dyz orbitals and close to axial symmetry of the ligands coordinating this heme. This is in a good correspondence to the near perpendicular mutual orientation of axial histidine planes of heme IV, deduced from the patterns of

13

C NMR paramagnetic shifts

46

and

known from PpcA structure, Table 1. From the analysis of the EPR spectrum we can estimate the interdependence of ligand-field parameters in PpcA62, 80. As was shown for monoheme and tetraheme cytochromes c3

81

, the

amplitude of EPR signal at gy principal value increased (while at gx decreased), relative to the amplitude of gz peak with the strengthening of correlation between the V and ∆ parameters. The ratio between these signals in the PpcA EPR spectrum, in particular for heme III, where the amplitude of gy principal value is lower than gz, indicates opposite tendency and, thus, the lack of strong correlation between the V and ∆ parameters. The simulations for heme I and heme IV, Figure 6, showing HALS signals with low-field amplitude higher than other principal g values, also illustrate a similar propensity. For PpcA this observation can be attributed to relatively independent variation of ligand field parameters associated with individual hemes. When comparing the triheme cytochrome c7 to tetraheme c3-cytochromes, which have parameters that are more correlated

81

, we have to point out that the heme core arrangement is less compact in

cytochromes c7 than in cytochromes c3, as was evident in the increase of Fe–Fe distances between the hemes

22, 24

. Therefore, we presume that diminishing correlation between ligand

field parameters is the consequence of the absence heme II and possibly ensuing more adaptable coordination geometries in PpcA.

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Biochemistry

Figure 6. Simulated EPR spectra of individual low spin hemes from PpcA cytochrome with corresponding structures. The 5th histidine ligand from CXXCH binding motif located at the top of the porphyrin planes.

Magnetic interactions. For redox proteins with multiple paramagnetic cofactors, the possibility of resolving inter-cofactor magnetic couplings is of particular interest 82. There are two types of magnetic interactions important for these systems: anisotropic dipole-dipole spin interaction, decreasing with distance proportionally to 1/r3, and (isotropic) exchange interaction, demonstrating an exponential decay with increasing distance. The interactions between hemes in PpcA can be estimated from the reported structures of this cytochrome in ferric state

22, 35

. The

shortest distance separating the iron centers is 11.6 Å between heme I and heme III and

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comparable distance 12.6 Å found for the heme III and heme IV pair. An even larger distance separates heme I and IV, i.e. 18.5 Å according to the solution structure

35

. Assuming the

minimum separation of 11.6 Å, the dipolar coupling for two point dipoles can be estimated as ca. 1.2 mT. Taking into account that the narrowest line in our EPR spectrum has a line width of > 7.0 mT, it is not possible to observe this dipole-dipole magnetic interaction between hemes. Having a separation of about 12 Å between the heme centers makes it is difficult to know a priori if a substantial exchange coupling was present. The estimation of the exchange coupling as a function of distance is complex, since it is also strongly depending on the intervening medium. For this distance it can be prominent for the directly bound prosthetic groups, or weak if there are no conjugated bond paths between the spin-carrying cofactors and exchange interaction is primarily through the space

83, 84

. Significantly, line broadening or splitting due to

exchange interactions are not detected for the heme cofactors in PpcA, which is an indication of weak or absent magnetic spin couplings. This magnetic independence is similar to tetraheme cytochromes of sulfate reducing microorganisms with comparable iron-to-iron distances, where effect of magnetic interactions is very weak due to explicit arrangements of hemes in diheme clusters

81, 82

. By contrast, EPR spectra of redox proteins with prominent heme-heme magnetic

coupling have complicated multi-peak spectra, as observed in tetraheme cytochrome c-554 and multiheme hydroxylamine oxidoreductase

87, 88

85, 86

of the ammonia oxidizing system in

Nitrosomonas europeae. At the same time, along with characteristic signals of bis-His coordinated hemes, two additional weak signals around g=2.03 and 2.63 were observed in the EPR spectrum of PpcA. While the first signal could arise from damaged protein or an impurity admixture in the sample, the origin of the second positive peak was not clear. We detected this peak in all our PpcA samples with consistent relative intensity. The significant solvent exposures of the PpcA hemes and opportunity for close heme-heme contacts and interactions in protein aggregates suggests the possibility that this minor signal could arise in a small fraction of the protein where multimer complexes might be partially induced by the high concentration of cytochrome used for EPR experiments. A possible alternative explanation could be the presence of conformational substates in the protein. The variations in the axial ligand to Fe distances could result in the emergence of type III EPR spectra having (dxz, dyz)4 dxy1 electronic configuration with gx around 2.6. On-going measurement are attempting to identify the source for this signal.

22


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Biochemistry

CONCLUSION EPR spectroscopy has been used to resolve the EPR signatures of three individual hemes within multiheme PpcA cytochrome. The assignment of each EPR spectrum to the particular heme was implemented referring to the solution NMR and X-ray crystal structures. Electronic configuration of the Fe(III) and its ligation mode, as well as relative energy levels of the Fe(III) 3d orbitals for individual hemes, were determined based on the analysis of the g-factors. EPR analysis has been supplemented with the investigation of porphyrin macrocycles by 1H NMR of heme methyl substituents to ensure unambiguous assignment of highly anisotropic low spin signals to individual hemes. It was shown that the magnetic interactions between hemes are minimal in PpcA, similar to the c3 family of tetraheme cytochromes. ASSOCIATED CONTENT

Supporting information This material is available free of charge via the Internet at http://pubs.acs.org Description of the rationale for ligand-field parameters calculations. Temperature dependence of the triheme cytochrome PpcA CW X-band EPR spectra. Plot of correspondence of paramagnetic shifts to orientation angle for heme III. Relative energy diagrams of individual hemes in PpcA.

ACKNOWLEDGEMENTS

We acknowledge the NMR Spectroscopy Laboratory in the Chemical Sciences and Engineering Division of Argonne National Laboratory for the use of instrumentation. We thank Dr. John Muntean and Prof. Carlos Salgueiro for assistance and guidance in acquiring of NMR spectra. We are grateful to Dr. N.V. Shokhirev for advices in analysis of NMR data and results received using ShiftPatterns program. The contribution of Prof. Marianne Schiffer in this research and many useful discussions is highly appreciated. FUNDING 23


Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The material is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences, under Contract DE-AC02-06CH11357 at Argonne National Laboratory.

ABBREVIATIONS PpcA, periplasmic cytochrome A; EPR, electron paramagnetic resonance; NMR, nuclear magnetic resonance; HALS, highly axial low spin.

REFERENCES

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EPR Characterization of the Triheme Cytochrome from Geobacter sulfurreducens Nina Ponomarenko*, Jens Niklas, P. Raj Pokkuluri, Oleg Poluektov, David M. Tiede*

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