Unpaired Electron Spin Density Distribution across Reduced [2Fe-2S

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

pubs.acs.org/IC

Unpaired Electron Spin Density Distribution across Reduced [2Fe2S] Cluster Ligands by 13Cβ‑Cysteine Labeling Alexander T. Taguchi,†,⊥ Yoshiharu Miyajima-Nakano,† Risako Fukazawa,† Myat T. Lin,# Amgalanbaatar Baldansuren,∥,∇ Robert B. Gennis,‡ Kazuya Hasegawa,§ Takashi Kumasaka,§ Sergei A. Dikanov,*,∥ and Toshio Iwasaki*,† †

Department of Biochemistry and Molecular Biology, Nippon Medical School, Sendagi, Tokyo 113-8602, Japan Department of Biochemistry, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States § Japan Synchrotron Radiation Research Institute (SPring-8/JASRI), Sayo, Hyogo 679-5198, Japan ∥ Department of Veterinary Clinical Medicine, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States ‡

S Supporting Information *

ABSTRACT: Iron−sulfur clusters are one of the most versatile and ancient classes of redox mediators in biology. The roles that these metal centers take on are predominantly determined by the number and types of coordinating ligands (typically cysteine and histidine) that modify the electronic structure of the cluster. Here we map the spin density distribution onto the cysteine ligands for the three major classes of the protein-bound, reduced [2Fe-2S](His)n(Cys)4−n (n = 0, 1, 2) cluster by selective cysteine-13Cβ isotope labeling. The spin distribution is highly asymmetric in all three systems and delocalizes further along the reduced Fe2+ ligands than the nonreducible Fe3+ ligands for all clusters studied. The preferential spin transfer onto the chemically reactive Fe2+ ligands is consistent with the structural concept that the orientation of the cluster in proteins is not arbitrarily decided, but rather is optimized such that it is likely to facilitate better electronic coupling with redox partners. The resolution of all cysteine-13Cβ hyperfine couplings and their assignments provides a measure of the relative covalencies of the metal−thiolate bonds not readily available to other techniques.

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provide a firm basis for the testing and further development of the theoretical approaches.6,7 Iron−sulfur clusters are involved in a multitude of ET reactions in biology. Their coordinating ligands primarily define the redox chemistry and electronic structure of the cluster, which is further tuned by factors such as hydrogen bonds.8−10 In this work, the 2D pulsed electron paramagnetic resonance (EPR) technique HYSCORE (hyperfine sublevel correlation) is performed on selectively Cys-13Cβ-labeled [2Fe2S](His)n(Cys)4−n (n = 0, 1, 2) proteins reduced in the S = 1/ 2 spin state in order to determine the hyperfine (HF) interactions with the nearby Cys-13Cβ′s. After integrating these results with previously reported HF couplings for the remote nitrogens of the histidine ligands, a detailed map of the unpaired s-spin density distribution in the immediate cluster environment relevant to biological ET is constructed. The spin distribution is highly asymmetric in all three systems and delocalizes further along the reduced Fe2+ ligands than the nonreducible Fe3+ ligands for all clusters studied.

INTRODUCTION Electron transfer (ET) between redox active cofactors and their biological partners depends critically on the extent of electron orbital overlap. X-ray crystal structure analysis has demonstrated that electrons are rapidly shuttled from one redox center to another by controlling the relative distances, redox potentials, and intervening protein environment.1 The proximity of the redox centers is the primary factor in facilitating efficient ET, which can be modeled by the edge-toedge distance between the cofactors, or by more sophisticated models using the distribution of electron spin density as a probe in quantum mechanics/molecular mechanics calculations for mapping out potential ET pathways.2 Hence, the distribution of electrons within the redox cofactor is directly linked to the mechanism of ET and may therefore provide a powerful handle on probing ET pathways and kinetics not available from X-ray crystal structures alone. For instance, joint nuclear magnetic resonance (NMR) and density functional theory work has correlated electron spin densities with ET rates.3 Quantum chemical calculations have also found that the pattern of unpaired spin delocalization may play an important role in facilitating ET.4,5 Although the electron distribution can be estimated computationally, experimental measurements © XXXX American Chemical Society

Received: October 17, 2017

A

DOI: 10.1021/acs.inorgchem.7b02676 Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

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EXPERIMENTAL SECTION

Article

RESULTS AND DISCUSSION The archaeal Rieske-type [2Fe-2S](His)2(Cys)2 ferredoxin from Sulfolobus solfataricus (ARF, n = 2),12 the ISC-like [2Fe2S](Cys)4 ferredoxin from Pseudomonas putida (FdxB, n = 0),15 and the thermophile mitoNEET [2Fe-2S](His)1(Cys)3 homologue from Thermus thermophilus (TthNEET, n = 1)16 are used as representative model proteins (Figure 1). These

Construction of a New E. coli C43(DE3) Cysteine Auxotrophic Expression Host Strain YM154. In order to sitespecifically introduce isotopic labels into the protein environment near biological paramagnetic metallo-cofactors for high-resolution pulsed EPR and NMR study, we have recently reported the construction of a set of cost-effective, high-yield auxotrophs in the commonly used E. coli expression host strains C43(DE3) and BL21(DE3).11 These include the cysteine auxotrophic strain YM138 having a deletion of the cysE gene from the chromosome of E. coli C43(DE3) with the following set of polymerase chain reaction (PCR) primers: cysE.UpF, AGG GGG CAG TAT GCT AAA CAT CGT AC; cysE.UpR, GGA ATA GGA ACT AAG GAG GAT ATT CAT ATG TGT CCT GTG ATC GTG CCG GAT GC; cysE.DownF, CTT CGA AGC AGC TCC AGC CTA CAC TGC TTA CTC CAC ACG ATG AGA TAA TGA CC; cysE.DownR, TGA AAA ACG TCA TTG CCA TTG GTG CG. To facilitate high-level expression of the foreign genes with rare codons coding for metalloenzymes from thermophilic archaea and bacteria,12 we then incorporated a pACYCbased plasmid harboring tRNA genes (argU, ileY, and leuW) for the E. coli rare codons (Agilent Technologies) into the YM138 strain. The resultant new C43(DE3) cysteine auxotrophic expression host strain YM154 was used for the site-specific isotope labeling of the Fe−S proteins in this work. The knockout of the chromosomal cysE gene in the host cells was verified by PCR prior to use. Selective Cysteine 13C Isotope Labeling. Selective Cys-13Cβ isotope labeling of Sulfolobus solfataricus ARF in the 15N-protein background, Pseudomonas putida FdxB in the 15N-protein background, and Thermus thermophilus TthNEET in the 14N(natural abundance)protein background was achieved with YM154 grown in the presence of the labeled cysteine. Protein expression and purification protocols are described in detail in the Supporting Information. HYSCORE Experiments. Pulsed EPR experiments were carried out at 10 K for ARF, 20 K for FdxB, and 12 K for TthNEET using an X-band Bruker ELEXSYS E580 spectrometer equipped with an Oxford CF 935 cryostat. Two-dimensional, four-pulse hyperfine sublevel correlation spectroscopy (HYSCORE, π/2−τ−π/2−t1−π− t2−π/2−τ−echo)13 was employed with appropriate phase-cycling schemes to eliminate unwanted features from the experimental echo envelopes. The intensity of the echo after the fourth pulse was measured with t2 and t1 varied and constant τ. The length of a π/2 pulse was nominally 16 ns and a π pulse 32 ns. HYSCORE data were collected in the form of 2D time-domain patterns containing 256 × 256 points with steps of 32 ns. Spectral processing of ESEEM patterns, including subtraction of the relaxation decay (fitting by polynomials of 3−4 degree), apodization (Hamming window), zero filling, and fast Fourier transformation (FT), was performed using the Bruker WIN-EPR software. Spectral Simulations. HYSCORE simulations were performed with EasySpin v4.5.514 in Matlab R2014b. For orientation-selective HYSCORE spectra, a careful determination of the effective excitation bandwidth at each field position is important for accurate simulations. This was done by considering the broadening of the EPR spectrum and the selectivity of the microwave pulses, which are the main contributors to the excitation bandwidth as outlined by EasySpin. Of these, the EPR broadening was determined by simulating the X-band EPR spectra. The simulation parameters for ARF were gz = 2.022, gy = 1.901, and gx = 1.804 with line broadenings (modeled using the EasySpin H-Strain parameter) of 55, 100, and 253 MHz, respectively. For FdxB they were gz = 2.020, gy = 1.936, and gx = 1.934 with line broadenings 34, 37, and 69 MHz, respectively. Finally, for TthNEET the g-values were gz = 2.009, gy = 1.932, and gx = 1.896, and the line broadenings were estimated from the HYSCORE simulations as 50, 80, and 60 MHz, respectively (a reliable determination from EPR simulations was not possible due to the presence of features in the spectrum arising from spin−spin interactions between the two reduced clusters of the protein dimer). The excitation bandwidth of the microwave pulses was optimized during the simulation process.

Figure 1. Crystal structures (left)12,15,16 and a zoomed-in view of the upper-left region of the 13C HYSCORE spectra in contour presentation (right) for ARF measured at the magnetic field corresponding to the gx value (A), FdxB at g⊥ (B), and TthNEET at gy (C). The Cys 13Cβ-carbons are shown in green, and their corresponding cross-ridges in the HYSCORE spectra are marked as 1C−4C. The dashed antidiagonal lines in these spectra are defined by να + νβ = 2ν13C.

proteins were studied by pulsed EPR in the reduced state of the cluster, where the Fe3+ ion coordinated to the histidine ligand(s) is reduced to Fe2+.17,18 For FdxB with complete cysteinyl coordination (n = 0), the reduced Fe2+ site has been assigned as the one closer to the protein surface by electron− nuclear double resonance (ENDOR) analysis of the proton HF interactions,15 as in the cases reported for the structurally related n = 0 homologues.19−21 For the 13Cβ(Cys)-labeled ARF, the HYSCORE spectrum (Figure 1A) reveals 13Cβ crossfeatures from the Cys42 and Cys61 ligands coordinated at the nonreducible Fe3+ site. The two 13Cβ HF interactions are unresolved, but the presence of combination peaks with double frequency coordinates at very low contour levels confirms the assignment of two overlapping cross-ridges (Figure S4). The overall shape of the 13C cross-ridges is elongated and shifted off of the antidiagonal dashed line defined by να + νβ = 2ν13C, characteristic of 13C tensors with significant HF anisotropy.22 The FdxB cluster has a complete cysteinyl coordination, so HYSCORE spectra are expected to produce up to four 13CβCys cross-features. Four ridges were resolved (Figure 1B), with B


Article

Inorganic Chemistry two anisotropically elongated cross-ridges similar to that observed for ARF (1C and 2C), and two additional components showing weak anisotropy (3C and 4C). Out of all three model proteins, TthNEET exhibits the largest asymmetry in the ligand type arrangement, with two Cys ligands at the nonreducible Fe3+ site and one Cys ligand at the reduced Fe2+ site (Figure 1C). The 13Cβ-Cys HYSCORE spectra show two anisotropic cross-ridges (1C and 2C) and a third isotropic cross-peak 3C at low contour levels. An important advantage of the 2D spectroscopic approach demonstrated here is the resolution of cross-ridges from nuclei with different degrees of HF anisotropy. These features would otherwise be masked by more intensive ridges in 1D spectra. A full analysis of the 13Cβ-Cys HYSCORE spectra acquired at multiple orientations spanning the full g-tensor for each protein was performed, involving linear regressions of the cross-ridges in (ν1)2 versus (ν2)2 coordinates22 (see SquaredFrequency Analysis, Figures S1−S3, and Table S1 in Supporting Information) and spectral simulations (Figures S4−S7). The 13C HF tensors are grouped by their value of T, where 1C and 2C exhibit a high degree of anisotropy (1.2−1.4 MHz) and 3C and 4C are less anisotropic (0.3−0.5 MHz) (Table 1). The range of these 13Cβ HF anisotropies can be Table 1.

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ligands. Comparison of these two scenarios with the values of T in Table 1 allows the clear assignment of 1C and 2C to Fe3+ ligands, and 3C and 4C to Fe2+ ligands. For TthNEET, 3C is thus immediately assignable to Cys48 at the Fe2+ site. For the assignments of the 13C tensors to particular nuclei, an analysis of the simulated Euler angles (α, β, γ) within the gtensor reference frame must be considered. Only for FdxB has the g-tensor orientation been determined,15 and therefore, the discussion will be limited to this system. For the point−dipole interaction between the 13Cβ and its nearest iron, the A∥ component of the HF tensor is expected to lie along the line connecting the two atoms. The Euler angle β (angle between A∥ and gz) predicted from the FdxB PDB coordinates (3AH7.pdb)15 is 11° for Cys50 and 82° for Cys86. These values only agree with the assignment of 1C to Cys86 and 2C to Cys50 (Table 1). The Fe2+ coordinated cysteines are not considered here, because the Euler angles for these 13C tensors (3C and 4C) could not be accurately determined. The 13C isotropic HF couplings in Table 1 are directly related to the degree of unpaired spin delocalization onto the cysteine ligands of the cluster. By combining this data with the His-Nε HF couplings for ARF25 and TthNEET (Figure S7B), maps of the s-spin density distribution across all ligands of the reduced [2Fe-2S](His)n(Cys)4−n (n = 0, 1, 2) clusters were constructed (Figure 2). Among these three systems, the two

C HF Tensors from HYSCORE Simulations

HFCCa

1C

2C

a (MHz) T (MHz) δ (MHz) α (deg) β (deg) γ (deg)

0.8 1.3 0.1 −35 −59 89

± ± ± ± ± ±

0.2 0.1 0.1 10 9 33


1.2 1.2 0.2 54 27 64

± ± ± ± ± ±

0.1 0.1 0.1 16 10 80


1.1 1.3 0.2 40 81 33

± ± ± ± ± ±

0.2 0.1 0.1 180 12 62

ARF 0.4 ± 0.2 1.3 ± 0.1 0.2 ± 0.1 25 ± 22 53 ± 6 0 ± 14 TthNEET −0.1 ± 0.2 1.4 ± 0.1 0.2 ± 0.2 53 ± 12 −58 ± 14 59 ± 21 FdxB −0.2 ± 0.2 1.2 ± 0.1 0.2 ± 0.2 −30 ± 180 −19 ± 11 82 ± 180

3Cb

4Cb

3.0 ± 0.2 0.3 ± 0.2 0 0 20 0 2.8 ± 0.1 0.5 ± 0.1 0 0 50 0

Figure 2. Maps of the outer-shell s-orbital electron spin density distribution onto the ligands of the reduced ARF (left), FdxB (middle), and TthNEET (right) clusters. Positive and negative electron spin is shown in blue and red, respectively. Green indicates the sign is unknown. The areas of the circles show the relative spin populations.

1.8 ± 0.2 0.5 ± 0.2 0 0 −60 0

Fe3+ ligands in ARF are most symmetrically positioned with respect to the cluster (Figure 1). Even in this case, the His-Nε spin populations at the reduced Fe2+ site of ARF are, on average, almost twice larger in magnitude than the corresponding 13Cβ-Cys values at the nonreducible Fe3+ site, despite the His-Nε being one bond further away from the cluster than Cys-Cβ. This would suggest that the transfer of unpaired spin extends more along the Fe2+ ligands than those of Fe3+ in ARF. The spin density is approximately evenly distributed between the two Cys ligands, reflecting the symmetrical orientations of Cys42 and Cys61 (Fe2+−Fe3+− Sγ−13Cβ dihedral angle θ values are +117° and +113°, respectively)11 relative to the cluster. The two other systems exhibit an asymmetric arrangement of two Fe3+ ligands with respect to the cluster (Figure 1). For FdxB, the magnitude of electron spin transferred onto Cys-Cβ at the Fe2+ site is, on average, more than 5 times larger than that transferred to the Fe3+ side (Table 2), in line with the ARF data. However, unlike ARF, the Cys50 and Cys86 Cβ spin

a HF coupling constants. a, T, and δ are the isotropic, anisotropic, and rhombic constants, respectively. Euler angles α, β, and γ describe the rotations that bring the g-tensor into the HF tensor eigenframe. bDue to the low intensity of 3C and 4C, axial HF tensors were assumed (δ = 0) and the Euler angles were adjusted manually. The errors reported for a and T were thus estimated by eye.

explained by the point−dipole model for the magnetic interaction between the 13C nucleus and the Fe spin density. By modeling the Fe3+ and Fe2+ spin populations with the normalized coefficients DS determined in similar protein systems (+1.60 to +1.85 for Fe3+ and −0.55 to −1.24 for Fe2+),15,21,23,24 the dipolar contribution to the anisotropic constant for a coordinated Cys-Cβ is estimated to be 0.9−1.0 MHz for the Fe3+ ligands and 0.3−0.7 MHz for the Fe2+ C


Article

Inorganic Chemistry Table 2. Cys-13Cβ s-Orbital Spin Populations (×10−4)a 1C (Fe3+) 2C (Fe3+) 3C (Fe2+) 4C (Fe2+) His-Nε (Fe2+)

ARF

TthNEET

FdxB

1.4 ± 0.2 0.6 ± 0.2

2.2 ± 0.2 −0.2 ± 0.3 |7.9| ± 0.5

1.9 ± 0.3 −0.4 ± 0.4 |7.4| ± 0.3 |4.7| ± 0.5

∼|1.8|25

∼|1.9|b

of FdxB would first require an assignment of the HF tensors to specific nuclei.

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CONCLUSIONS In summary, we have developed the tools to characterize the electronic structure of the biological [2Fe-2S](His)n(Cys)4−n (n = 0, 1, 2) clusters in the ground state by measuring the distribution of unpaired s-spin density on the cysteine and histidine ligands. Our results have identified a consistent trend where a significantly larger amount of unpaired spin is transferred to the second coordination sphere Fe2+ site ligands than that of Fe3+, despite the lower S = 2 spin of Fe2+ than S = 5/2 of Fe3+. The preferred delocalization of the electron spin along the Fe 2+ site ligands correlates well with ET directionality of these proteins; redox chemistry is exclusively catalyzed at the reducible Fe2+/Fe3+ site of the cluster, maximizing the electronic coupling with redox partners.15,20,21,24,30−35 The amino-acid-selective isotopic labeling strategy11 and 2D spectroscopy allow separation and assignments of the resonances from Cys-13Cβ located at the second coordination sphere of different irons, not possible from uniform 13C labeling and 1D experiments.36 In spite of the wide application of NMR to quantify electron spin distributions in paramagnetic systems,37,38 Cys-13Cβ nuclei around the paramagnetic Fe−S cluster are located too close to the irons and are often not observed experimentally.39,40 The methodology used in this work opens the way for direct determination of the isotropic and anisotropic couplings for 13 Cβ-Cys in paramagnetic clusters complementary to the paramagnetic NMR approach. In conjunction with DFT computations it is expected to be applicable for rapid and robust characterizations of the spin density distribution onto the ligands of other clusters and metallocomplexes, critical to a quantum mechanical understanding of the chemistry.

a

s-Orbital spin populations were estimated by normalizing the isotropic constants a to the magnetic moment of the nearest coordinating Fe (see Supporting Information),26 and then dividing this number by the appropriate atomic HF constant (a = 3777 MHz for 13C and a = 1811 MHz for 14N).27 The absolute signs of the spin populations were determined using the known sign of the Fe3+ spin (positive) as a reference for 1C and 2C. The low HF anisotropy for the other HF tensors prevents a similar analysis for the Fe2+ ligands. b Figure S7B in Supporting Information.

populations are very different, probably due to the different geometries of these ligands with respect to the cluster (θ of +81° for Cys50 and −117° for Cys86) constrained by the surrounding polypeptide chain.15 Different geometries of the Fe2+ ligands (θ of +167° for Cys41 and +127° for Cys47) may also have contributed to an imbalanced spin transfer to these nuclei (Table 2). In TthNEET, the Fe2+/Fe3+ ligands are also asymmetrically positioned with respect to the reduced cluster (θ of +9° for Cys37, −90° for Cys39, and −20° for Cys48),16 and as a result the pattern of spin transfer to the Fe3+ cysteine ligands is highly asymmetric, like the case for FdxB (Figure 2). The 13Cβ spin population at the Fe2+ cysteine was found to be almost 7 times larger than the average value at the cysteines of the Fe3+ site, again similar to what is observed for FdxB. These results suggest that the local structural asymmetry enforced by the surrounding polypeptide chain, rather than the types of the cluster ligands, regulates the electron spin distribution across Cys-Cβ nuclei at the reduced cluster site (Table 2 and Figure 2). In [2Fe-2S](His)n(Cys)4−n (n = 0, 1, 2) proteins, the fundamental redox chemical properties of the cluster are primarily defined by the covalency of the Fe−ligand interactions. This was first addressed by sulfur K-edge X-ray absorption spectroscopy, where the pre-edge feature was used to analyze the covalency of the average Fe3+−thiolate and Fe3+−sulfide bonds in the oxidized and reduced [2Fe2S](His)n(Cys)4−n (n = 0, 2) proteins.28,29 The present 13CβCys HYSCORE analysis of the reduced [2Fe-2S](His)n(Cys)4‑n (n = 0, 1, 2) proteins successfully resolves the covalent interactions of the cysteine ligands with the same iron for the first time. The electron spin population on Cys-Cβ corresponds to the degree of spin delocalization via orbital overlap, and should therefore provide a relative measure of the Sγ-Fe covalencies. In ARF, the electron spin populations on 13 Cβ of Cys42 and Cys61 were found to differ by only 0.8 × 10−4, suggesting slightly different covalencies of the two Sγ−Fe bonds. For the Fe3+ ligands of FdxB and TthNEET, where the ligand placement relative to the cluster is asymmetrical, the differences in spin populations are greater at 2.3 × 10−4 and 2.4 × 10−4, respectively. A similar situation applies to the Fe2+ ligands of FdxB where the difference in Cβ spin densities is 2.7 × 10−4. For the Fe3+ ligands of FdxB, the greater spin transfer to the Cys86-Cβ would suggest that Cys86 has the stronger of the two Sγ−Fe covalent interactions. A similar residue-specific analysis for the Fe3+ ligands of TthNEET and the Fe2+ ligands

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.7b02676. Materials and HYSCORE methods (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Alexander T. Taguchi: 0000-0002-5940-5948 Robert B. Gennis: 0000-0002-3805-6945 Sergei A. Dikanov: 0000-0003-2610-6439 Present Addresses ⊥

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, United States. # Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14850, United States. ∇ School of Chemistry and EPRSRC National EPR Facility, University of Manchester, Manchester, M13 9PL, United Kingdom. Author Contributions

The manuscript was written by contributions of all authors. All authors approve of the final version of the manuscript. D


Article

Inorganic Chemistry Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This investigation was supported in part by the International Collaborations in Chemistry Grant from JSPS (T.I.) and NSF (CHE-1026541 to S.A.D.), the JSPS Grant-in-Aid 24659202 (T.I.), the DE-FG02-87ER13716 (R.B.G.) and DE-FG0208ER15960 (S.A.D., pulsed EPR work) Grants from the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Sciences, U.S. DOE, and NIGMS Roadmap Initiative (R01GM075937). A.T.T. gratefully acknowledges support as a JSPS Postdoctoral Fellow (P14415).

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Unpaired Electron Spin Density Distribution across Reduced [2Fe-2S

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