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Article 13

Determination of the Complete Spin Density Distribution in C-Labeled Protein-Bound Radical Intermediates Using Advanced 2D Electron Paramagnetic Resonance Spectroscopy and Density Functional Theory Alexander T. Taguchi, Patrick J. O'Malley, Colin A. Wraight, and Sergei A. Dikanov J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b10036 • Publication Date (Web): 16 Oct 2017 Downloaded from http://pubs.acs.org on October 17, 2017

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The Journal of Physical Chemistry

Determination of the Complete Spin Density Distribution in 13C-Labeled Protein-Bound Radical Intermediates Using Advanced 2D Electron Paramagnetic Resonance Spectroscopy and Density Functional Theory Alexander T. Taguchi,*,#,‡,@ Patrick J. O’Malley,*,& Colin A. Wraight,#,§,∆ and Sergei A. Dikanov*,‡ #

Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States ‡ Department of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States & School of Chemistry, The University of Manchester, Manchester M13 9PL, U.K. § Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States *To whom correspondence should be addressed: A.T.T.: email, [email protected]. S.A.D.: email, [email protected]; phone, (217)300-2209. P.J.O.: email, patrick.o’[email protected]; phone, 00441612004536. Present Address: @ Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, United States ∆ deceased on July 10, 2014

ABSTRACT: Determining the complete electron spin density distribution for protein-bound radicals, even with advanced pulsed electron paramagnetic resonance (EPR) methods, is a formidable task. Here we present a strategy to overcome this problem combining multifrequency HYSCORE and ENDOR measurements on site-specifically 13C-labeled samples with 1

ACS Paragon Plus Environment


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DFT calculations on model systems. As a demonstration of this approach, pulsed EPR experiments are performed on the primary QA and secondary QB ubisemiquinones of the photosynthetic reaction center from Rhodobacter sphaeroides

13

C-labeled at the ring and tail

positions. Despite the large number of nuclei interacting with the unpaired electron in these samples, two-dimensional X- and Q-band HYSCORE and orientation selective Q-band ENDOR resolve and allow for a characterization of the eight expected

13

C resonances from significantly

different hyperfine tensors for both semiquinones. From these results we construct, for the first time, the most complete experimentally determined maps of the s- and pπ-orbital spin density distributions for any protein organic cofactor radical to date. This work lays a foundation for understanding the relationship between the electronic structure of semiquinones and their functional properties, and introduces new techniques for mapping out the spin density distribution that are readily applicable to other systems.

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Introduction The spin density distribution of an organic or biological radical provides a unique signature of its electronic structure, in particular the electron density of the singly occupied molecular orbital (SOMO). Electron paramagnetic resonance (EPR), and especially pulsed EPR, is the technique of choice for probing this spin density distribution by measuring the magnetic interaction strength with nearby nuclear spins.1 Recent developments with auxotrophic strains for amino acid-specific isotope labeling2, protein sequence4,

7

3

and incorporation of unnatural amino acids into the

have provided EPR spectroscopists with powerful tools to probe the

magnetic interactions between the electron and nuclear spins at a level of atomic specificity previously unachievable. Despite these biochemical advancements in EPR sample preparation, the electron spin density distribution has yet to be completely characterized for any bioorganic radical cofactor.8, 9 The joint application of pulsed EPR and Density Functional Theory (DFT) calculations can provide high-resolution mechanistic insight into the functional and structural properties of radical cofactors unavailable from X-ray crystal structures alone. DFT offers a robust quantum mechanical modeling method for relating the spin density distribution to molecular structure, from which the experimentally measurable hyperfine interaction parameters can be calculated. This interaction describes the magnetic coupling between the electron and nuclear spins, and directly reflects how the electron spin is distributed in the molecule. By comparing the experimental parameters with quantum mechanical calculations, resonance assignments can be made to particular atoms. This leads to a model of the spin density distribution that defines the redox properties of the radical, elucidates its high-resolution local structure, and contains details of how it is electronically coupled to the protein environment. This strategy has been applied 3



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with varying degrees of success to unravel the molecular structure and function for various spin systems.10-16 In many of these studies, the joint collaboration between pulsed EPR and DFT calculations has been found necessary to provide a full interpretation of the experimental data, where the correctness of the calculated structural model depends upon how well it reproduces the experimentally observed electron spin density distribution. However, for no protein-bound organic radical cofactor has the spin density distribution been completely experimentally determined through characterization of the hyperfine couplings with all atoms types (such as with natural abundance 1H and 14N magnetic nuclei or via isotopic enrichment with 13C, 15N and 17

O labeling). As a result, validation of DFT models relies upon agreement with experimentally

measurable hyperfine parameters for nuclei(us) of one or several substituents that act as representative probes of the overall spin density distribution. In this study, we develop a strategy for characterizing the electron spin density distribution using the semiquinone (SQ) radical of ubiquinone, one of the most prevalent redox mediators in Nature. Its ability to reversibly accept one or two electrons makes it a critical component in energy transduction pathways of photosynthesis and respiration, as well as in regulation of other aspects of cellular metabolism.17 In the photosynthetic reaction center (RC) from Rhodobacter sphaeroides, ubiquinone plays an essential role in the conversion of light energy into a transmembrane charge separation.18-21 Upon light excitation, an electron is transported across the membrane to the QA site, forming the anionic semiquinone radical QA(SQA). SQA cannot be reduced further and acts as a one-electron gate that shuttles electrons one at a time to the QB site located on the opposite side of a non-heme iron (Figure 1). Unlike QA, QB is capable of accepting two electrons sequentially from QA, first forming the QB- semiquinone 4


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(SQB) followed by full reduction to quinol (QBH2).20, 21 The unique redox properties of SQA and SQB, and the ability to generate the semiquinones with ~100% quantum efficiencies,22 makes the RC an excellent candidate for studying the relationship between SQ electronic structure and function. Developing the biochemical and spectroscopic methodologies to overcome these issues using SQA and SQB in the RC as a model system is the major focus of this work.

Figure 1. Symmetric locations of the QA and QB sites about the non-heme iron (green) in pdb entry 1DV3. The quinone ring carbons are numbered as referenced in the text, and only the first (CH2) and second (CH) carbons of the isoprenoid tails are shown extending from the C6 position. Differences in the number and strengths of the hydrogen bonds between the semiquinones and their surrounding residues give rise to unique spin density distributions for SQA and SQB.

SQA and SQB samples are made with all quinone ring and isoprenoid tail carbons selectively 13C-labeled. Simulations and analyses of the X- and Q-band HYSCORE (Hyperfine Sublevel Correlation) and Q-band ENDOR (Electron-Nuclear Double Resonance) spectra resolve up to eight

13

C hyperfine tensors for each semiquinone. Six tensors with significant

hyperfine anisotropy are expected corresponding to ring carbons C1 through C6 (Figure 1). The remaining 13C couplings lack a strong anisotropic component and are associated with the nearest two carbons of the isoprenoid tail. The results of this study are then combined with previously 5



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reported 1H (5’-methyl, hydrogen bonds), 13C (methoxy, 5’-methyl, ring carbon), 17O (carbonyl), and

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N (H-bond donors) couplings to construct an extensive map of the s- and pπ-orbital spin

density distribution across the quinone ring carbons, their substituents, and the hydrogen bonds for SQA and SQB. A comprehensive understanding of the s- and pπ-orbital spin density distributions for these semiquinone systems will facilitate the creation of more reliable DFT models for SQA, SQB, and other semiquinones, provide a useful framework to calculate their light-induced electron transfer rates,18-21, 23 act as a platform for investigations of the spin density distribution in other organic radical cofactors, and advance general DFT development efforts.24

Experimental Section Sample Preparation. Previously, we prepared headgroup

13

C-methyl labeled ubiquinone

for pulsed EPR studies of the RC.25 In the present work, we apply the reverse labeling approach where all carbons except the headgroup methyls are

13

C-labeled. For this purpose, ubiquinone

was biosynthesized in a strain of E. coli2 that is auxotrophic for eight amino acids, including methionine, which is the methyl donor in ubiquinone synthesis. This strain was grown on

13

C-

glucose (obtained from Cambridge Isotopes) as the carbon source in media supplemented with the necessary amino acids with natural abundance levels of

12

C. This growth scheme results in

ubiquinone where only the quinone ring and isoprenoid tail carbons are

13

C-labeled, as

confirmed by mass spectrometry. The ubiquinone was extracted in organic solvents and purified by TLC.26 The product of this biosynthesis in E. coli is UQ-8, rather than the native UQ-10 in Rb. sphaeroides, but no functional differences exist between them.27

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Reaction centers used in this study were isolated from a strain of Rb. sphaeroides expressing RCs with a histidine-tag on the M subunit.28 Cells were grown with

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N-ammonium

sulfate (obtained from Cambridge Isotopes) as the nitrogen source, to prevent possible peak overlap or cross-suppression effects of 14N on the 13C modulation.29 In order to remove the broad signal arising from the magnetic coupling of the semiquinone with the high spin Fe2+, the nonheme iron was biochemically replaced with diamagnetic Zn2+ according to the procedures outlined by Utschig et al.30 For SQA sample preparations, the quinones from both the QA and QB sites were extracted by the method of Okamura et al.,31 and were then replaced with the

13

C-

labeled ubiquinones. The SQA radical was generated by reduction with 8 mM Na-dithionite in semianaerobic conditions (continuous argon flow over the sample).32 For SQB sample preparations, only the QB site quinones were extracted and replaced with the 13C ubiquinone such that any contaminating SQA signal would not contribute to the 13C spectrum. As a consequence of this, CW EPR measurements showed indications of incomplete 13C enrichment (arising from either 12C-SQA,

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C-SQB, or both). SQB was generated by exposing the RCs to a single 532 nm

Nd:YAG laser pulse in the presence of a 3-fold excess of ferrocytochrome c (to quickly reduce the bacteriochlorophyll dimer after charge separation).32 Upon semiquinone formation, all samples were frozen promptly in liquid nitrogen. ESEEM and ENDOR Experiments. The instrumentation, pulse sequences, and spectral processing for X-band two-dimensional HYSCORE33 (π/2-τ-π/2-t1-π-t2-π/2-τ-echo) was as described previously.32 Q-band HYSCORE and ENDOR measurements were carried out on an Oxford CF 935 cryostat equipped with an EN 5107D2 resonator. Q-band HYSCORE pulse sequences and spectral processing were the same as for X-band HYSCORE, except that the π/2and π-pulse lengths were 28 and 56 ns, respectively, and the time domain data was collected as 7



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256 × 256 points in steps of 16 ns. Q-band pulsed ENDOR spectra were acquired using the Davies (π-t-π/2-τ-π-τ-echo) sequence with a radiofrequency π-pulse inserted during time interval t. The specifics of these experiments are described both in the text and in detail elsewhere.1 Powder

13

C ESEEM and ENDOR Spectra. The high-resolution pulsed EPR techniques

ESEEM and ENDOR make use of the paramagnetic properties of the SQ intermediate to probe its interactions with nearby magnetic nuclei of the protein, the aqueous solvent, and the quinone molecule itself. The isotropic and anisotropic hyperfine interactions with magnetic nuclei such as 13

C can provide detailed information on the electronic structure and geometry of the SQ in its

binding pocket.34, 35 For a hyperfine coupled 13C nucleus with nuclear spin I = ½, there are only two transitions with frequencies να and νβ, corresponding to the two different spin states mS = ±½ of the SQ electron spin in a constant applied magnetic field. The values of these frequencies depend on the vector sum of the applied and local magnetic fields induced at the nucleus by the isotropic and anisotropic hyperfine interactions with the electron spin. For a powder spectrum, the frequencies of the να and νβ transitions span the range between να(β)⊥ = |ν13C ± A⊥/2| and να(β)|| = |ν13C ± A||/2|

(1)

which correspond to the perpendicular and parallel orientations, respectively, of the axial hyperfine tensor. ν13C is the Zeeman frequency of 13C in the applied magnetic field, and A⊥ = |a − T| and A|| = |a + 2T| (where a and T are the isotropic and anisotropic hyperfine coupling constants, respectively). The full axial hyperfine tensor has principal components (a − T, a − T, a + 2T). The principal values for a rhombic tensor are AX = |a − T(1 + δ)|, AY = |a − T(1 − δ)|, and AZ = |a + 2T|, where δ is the rhombic parameter (which ranges in value from 0 to 1). Powder 8


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HYSCORE and ENDOR spectra of I = ½ nuclei reveal, in the form of cross-ridges or Pake patterns, respectively, the distribution of nuclear frequencies at all molecular orientations. In this work we use X- and Q-band pulsed EPR with microwave frequencies ~9.7 and ~34 GHz, respectively. The X-band EPR spectrum of the SQ in frozen solutions is a single line with unresolved g-tensor anisotropy. The g-tensor broadening is comparable to the excitation width by microwave pulses, so in the X-band experiment pulses can be considered as giving complete excitation of the EPR spectrum. Therefore, at this microwave frequency the ESEEM and ENDOR powder spectra exhibit nuclear frequencies from all orientations of the applied magnetic field relative to the hyperfine tensor principal axes. On the other hand, at Q-band the principal components of the SQ g-tensor are partially resolved, allowing for orientation selective measurements by exciting only one section of the EPR spectrum at a time. The combined knowledge from non-selective (X-band) and selective (Q-band) methods can provide complementary information on the principal values and directions of the 13C tensors. Squared-Frequency Analysis of HYSCORE. The ideal cross-peak shape in HYSCORE spectra is an arc-type ridge between the points (να⊥, νβ⊥) and (να||, νβ||) located on the |να ± νβ| = 2ν13C lines. The shape of the ridge is described by the general equation να = (Qνβ2 + G)1/2

(2)

where Q and G are coefficients that are functions of a, T and ν13C.36 This lineshape transforms into a straight line segment in (να)2 vs (νβ)2 coordinates. The intensity of the cross-ridges is significantly suppressed at orientations around the principal directions (να⊥, νβ⊥) and (να||, νβ||), corresponding to orientations of the magnetic field along the A⊥ and A|| principal directions of the hyperfine tensor, respectively.37 Therefore, in HYSCORE spectra only the central part of the cross-ridge, which corresponds to orientations of the magnetic field substantially different from 9



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the principal directions, will possess observable intensity.37 The crossing points (να⊥, νβ⊥) and (να||, νβ||) can be determined by linear extrapolation of the observable parts in (ν1)2 vs (ν2)2 coordinates.36, 37 The coordinates of the crossing points can then be used to calculate A⊥ and A|| with Eq. 1. However, the assignments of the crossing points to A⊥ and A|| is unknown, resulting in two possible solutions. The correct solution of A⊥ and A|| can be determined by spectral simulations, ENDOR experiments, or comparison with DFT calculations. In the more complicated situation of a rhombic hyperfine tensor, cross-ridges connect the three points (ναX, νβX), (ναY, νβY), and (ναZ, νβZ) on the |να ± νβ| = 2ν13C lines, corresponding to the three principle directions of the tensor with principal values AX, AY, and AZ, respectively. Each pair of nuclear frequencies is described by ναi = |ν13C + |Ai|/2| and νβi = |ν13C – |Ai|/2| (where i = X, Y, and Z). The shapes of these ridges are described by the same Eq. 2, but with different coefficients Q and G that are now functions of a, T, δ, and ν13C. The relative intensities of the cross-ridges depend on the rhombicity parameter δ as: IZX:IZY:IXY = (3 + δ)3/2:(3 - δ)3/2:(2δ)3/2.37 For low values of δ, IXY is significantly smaller than IZX and IZY, and thus only two cross-ridges would be visible in the spectra. The three crossing points (ναi, νβi) on the |ν1 ± ν2| = 2ν13C line(s) can be obtained through linear regression of the observable parts of any two arcs in the (ν1)2 vs (ν2)2 representation of the spectrum. Spectral Simulations. ENDOR simulations were performed in Matlab R2014b with EasySpin package v5.0.22.38 All

13

C nuclei were modeled as rhombic hyperfine tensors with

principal values AX, AY, and AZ, and principal directions described by Euler angles α, β, and γ. The Euler angles are defined by EasySpin as the series of rotations that bring the g-tensor into the hyperfine tensor eigenframe.

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For orientation selective Q-band measurements, a determination of the effective excitation bandwidth at each field position is necessary to accurately simulate the spectra. This is done by considering the two major contributions to the excitation bandwidth in EasySpin: the broadening of the EPR spectrum and the selectivity of the microwave pulses. The EPR broadening parameters were estimated from simulation of the SQA Q-band CW spectrum (Figure S1). The SQB spectrum was not used for this purpose due to complications in generating a pure 13

C SQB sample described above. The excitation bandwidth of the pulses was approximated by

multiplying the inverse of the initial microwave π-pulse by two for Davies ENDOR. Additionally, the characteristic suppression of weak couplings in Davies ENDOR was taken into account by multiplying the simulations with the weighting function39

A(13 C ) 2 0.7 A(13 C ) 2 + ( ) 2 tp

(3)

which approaches zero and one for small and large values of the hyperfine coupling A(13C), respectively. Note that lengthening the first microwave π-pulse (tp) narrows the Lorentzian function, thereby reducing the suppression of weaker couplings. All other parameters were the same as those used in the experiments. DFT Calculations. DFT calculations were performed as previously described using the B3LYP functional.32 The EPR-II basis set was used for all atoms except Zn where 6-31g(d) was employed. All analyses were performed using the ORCA electronic structure program.40

Results X-band HYSCORE. X-band CW EPR measurements on

13

C-SQA show a strongly

broadened linewidth of 1.4 mT compared with the typical linewidth of ~0.8 mT for the natural 11



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abundance

12

C spectrum (Figure S2). The

13

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C hyperfine interactions responsible for nearly

doubling the spectral linewidth were investigated with X-band HYSCORE (Figure 2). The spectra were found to contain only a few recognizable 13C cross-peaks, despite that all ring and isoprenoid tail carbons (C1 through C6, and all carbons extending from C6 in Figure 1) are isotopically enriched in the sample. In the case of SQA, a unique spectral pattern of four straight ridges of similar intensity in both the (+,+) and (−,+) quadrants is observed, all with a common frequency in one dimension of 1.8 MHz. This pattern corresponds to a singularity in powder 1D ESEEM where all orientations contribute to the same frequency |3T/4| (= 1.8 MHz) in one electron spin manifold,41 allowing for an immediate calculation of the anisotropic component T = 2.4 MHz for this hyperfine tensor. The constraint |2a + T| = 4ν13C is expected to hold in this situation, where the isotropic constant is determined to be either a = -8.6 or 6.2 MHz. A very faint feature at (6, 1.5) MHz is also observed (asterisk in Figure 2), whose lineshape is suggestive of an additional tensor with weak anisotropy. Linear regression analysis of this cross-ridge in squared-frequency coordinates (Figure S3) provides an estimate of the hyperfine tensor with a = 4.6±0.6 or -5.2±0.6 MHz and T = 0.7±0.1 MHz. The broad feature at higher frequencies centered at ~(7, 7) MHz is far removed from the red dashed lines defined by |ν1 ± ν2| = 2(ν13C), indicating the presence of one or more strongly anisotropic

13

C tensors. However, the spectral resolution and signal intensity of this

region is too poor to allow for an estimate of the corresponding coupling constants.

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Figure 2. X-band 13C HYSCORE spectra for SQA (top) and SQB (bottom) in contour mode recorded with the time between the first and second pulses as τ = 136 ns. The red dashed lines are defined by |ν1 ± ν2| = 2ν13C. Experimental parameters: microwave frequency = 9.631 GHz (SQA) and 9.734 GHz (SQB), magnetic field = 343.4 mT (SQA) and 347.1 mT (SQB), temperature = 90 K.

For the SQB X-band HYSCORE spectrum, only a single doublet of

13

C cross-peaks is

observed, and appears in a similar location as in SQA (asterisk in Figure 2). An analysis of this cross-ridge in squared-frequency coordinates results in a = 4.0±0.1 or -4.5±0.1 MHz and T = 0.5±0.1 MHz (Figure S4). Otherwise, very faint 13C HYSCORE intensity near the noise level is detected between ~(-1, 8) and ~(-3, 10) MHz corresponding to the spread of hyperfine couplings ~9-13 MHz, but the poor quality of these features prevents any further analysis. Q-band HYSCORE. X-band HYSCORE failed to detect the expected number of

13

C

resonances for SQA and SQB, so samples were also probed by Q-band measurements. At Q-band, 13



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the microwave frequency and magnetic field are 3-4 times higher than at X-band, resulting in an increase in the

13

C Larmor frequency from 3.7 MHz (X-band) to 13 MHz (Q-band). As a

consequence of this, many of the expected 13C couplings fall into the weak coupling regime (ν13C > A(13C)/2) for most hyperfine tensor orientations. In the weak coupling limit, no intensity appears in the (−,+) quadrant of the HYSCORE spectrum. Instead, the cross-ridges are restricted to the (+,+) quadrant and have shallow modulation depths (because the cancellation condition ν13C ≈ A(13C)/2 is not satisfied), which is an important factor in avoiding internuclear crosssuppression effects.29

Figure 3. Q-band field swept two-pulse echo of SQA (top) and SQB (bottom) in samples reconstituted with ubiquinone 13C-labeled at the quinone ring and isoprenoid tail carbons. Field positions used for pulsed measurements are marked with red arrows for HYSCORE and yellow circles for Davies ENDOR. Experimental parameters: π/2-pulse length = 160 ns, time between first and second pulses τ = 820 ns, microwave frequency = 34.168 GHz, and temperature = 80 K.

Q-band HYSCORE measurements were performed at the magnetic fields corresponding to the principal values gX, gY, and gZ of the SQA and SQB g-tensors (Figure 3). The HYSCORE spectra obtained at gY are shown for both SQs in Figure 4 (see Figures S5-S10 for all gX, gY, and 14


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gZ spectra). In contrast to the X-band experiments, at Q-band many extended 13C cross-ridges are resolved.

Figure 4. Q-band 13C HYSCORE measurements at gY for SQA (top) and SQB (bottom) in contour mode presented as the sum of spectra recorded with the time between the first and second pulses as τ = 136 and 200 ns. The dashed line is defined by ν1 + ν2 = 2ν13C. Cross-ridge 1C’ for SQA, and the resolution of cross-ridges 1C’ and 2C’ for SQB, can only be observed at very low contour levels (Figures S5-S10). 3C and 4C of SQB are assigned as the overlap of two equivalent hyperfine tensors based on ENDOR simulations (Table 2). Experimental parameters: microwave frequency = 34.177 GHz (SQA) and 34.111 GHz (SQB), magnetic field = 1218.4 mT (SQA) and 1216.0 mT (SQB), temperature = 90 K. 15



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Figure 5. Linear regressions of the Q-band 13C HYSCORE cross-ridges for SQA (left) and SQB (right) in (ν1)2 vs (ν2)2 coordinates. Spectra were obtained as the sum of the spectra recorded at gX, gY and gZ with the time between the first and second pulses as τ = 136 and 200 ns. The curved line is defined by ν1 + ν2 = 2ν13C. Fits for the individual cross-ridges are provided in Supporting Information (Figures S11 and S12). Experimental parameters are the same as in Figure 4. The cross-ridges were analyzed in (ν1)2 vs (ν2)2 coordinates in order to extract the coupling constants for the interacting nuclei. The frequencies of the cross-ridges in the Q-band HYSCORE spectra recorded at gX and gZ were first recalculated to the same

13

frequency at gY (see “Normalization of HYSCORE frequencies to the same

13

C Larmor

C Larmor

Frequency” in Supporting Information) to allow for a simultaneous linear regression analysis of all spectra in (ν1)2 vs (ν2)2 coordinates (Figures S11 and S12).42 The resulting fits are shown overlaying the SQA and SQB Q-band HYSCORE spectra at gY in Figure 5. The two intersection points of the linear fits with the curved line defined by ν1 + ν2 = 2ν13C provide two possible solutions for the isotropic constant a (a1 and a2). The correct solution is designated as a1, as defined by the locations of the A⊥ singularities in the Q-band ENDOR spectra described below.

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A summary of the hyperfine coupling constants is provided in Table 1. 3C and 4C of SQB are interpreted as the overlap of two equivalent 13C tensors based on ENDOR simulations. We note that 5C of SQA agrees well with the singularity at 1.8 MHz in the X-band HYSCORE spectrum (a = -8.6 or 6.2 MHz and T = 2.4 MHz), but there is no match in the Q-band spectra for the weakly anisotropic features observed at X-band (asterisks in Figure 2).

Table 1. 13C hyperfine constants (MHz) from linear regression analysis of the Q-band HYSCORE spectra in (ν1)2 vs (ν2)2 coordinates.a

1C 1C’ 2C 2C’ 3Cb 4Cb 5C

a1

a2

SQA T

-0.4±0.2

-12.6±0.3

13.0±0.1

A⊥

A||

a1

-13.3±0.1

25.5±0.3

1.6

a2

SQB T

A⊥

A||

-14.8

13.2

-11.7

28.0

-2.2

-11.5

13.7

-15.9

25.1

1.6

-16.9

15.3

-13.7

32.2

6.2±0.1

-20.8±0.1

14.6±0.1

-8.4±0.1

35.5±0.1

4.1±0.1

-19.1±0.1

15.0±0.1

-10.9±0.1

34.2±0.1

6.3±0.1

-23.0±0.1

16.8±0.1

-10.5±0.1

39.8±0.1

3.3±0.1

-19.5±0.2

16.1±0.1

-12.8±0.1

35.6±0.2

0.0±0.1

-8.4±0.1

8.4±0.1

-8.4±0.1

16.8±0.1

-2.1±0.3

-4.4±0.3

6.5±0.1

-8.5±0.2

10.9±0.3

-0.2±0.3

-7.3±0.4

7.5±0.2

-7.7±0.1

14.8±0.4

-2.1±0.3

-4.4±0.3

6.5±0.1

-8.5±0.2

10.9±0.3

-8.4±0.1 6.3±0.3 2.2±0.2 -10.6±0.1 -4.1±0.3 -4.6±0.1 -0.7±0.1 5.3±0.1 -9.9±0.1 6.0±0.1 Linear regressions result in two possible solutions for a (a1 and a2) and one for T. The preferred solution for a is a1 based on Qband ENDOR simulations. The perpendicular (A⊥) and parallel (A||) principle values of the hyperfine tensor corresponding to the a1 and T pair are also shown. The errors were estimated by a comparison of the linear fits for cross-ridges on the opposite side of the diagonal. Therefore, errors could not be determined for nuclei where only one of the two cross-ridges is observed above the noise (1C’ from SQA and 1C and 1C’ from SQB). b 3C and 4C of SQB are assigned as the overlap of two equivalent hyperfine tensors based on ENDOR simulations (Table 2). a

Q-band ENDOR. Q-band Davies ENDOR measurements were performed to investigate the orientation dependence of the

13

C hyperfine tensors. Spectra were acquired at nine field

positions spanning gX, gY, and gZ (Figure 3). The SQA and SQB Q-band ENDOR spectra show a wealth of 13C peaks symmetrically located about ν13C ≈ 13 MHz (Figure 6). The only identifiable feature in the spectra that does not belong to the 13C couplings pertinent to this work is the SQA 15

N coupling at ~7 MHz.43 Due to both the presence of nitrogen coupling(s) and the nonuniform

power output of our radio-frequency generator in the lower frequency range of the 13C spectrum, 17



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Page 18 of 46

only the high frequency part of the spectrum above 13 MHz was fit to the simulations. For both semiquinones, high-intensity peaks at 17-20 MHz correspond to the perpendicular singularities (ν13C + |A⊥|/2) of the anisotropic tensors found from Q-band HYSCORE (Table 1). The disappearance of these features at orientations near gZ indicates that the principal axes of the A⊥ components of the tensors lie within the gX/gY plane, i.e., A|| is nearly collinear with the gZ axis. Sharp peak intensity that was undetected by Q-band HYSCORE (Figure 5) is observed in the 1417 MHz region of the ENDOR spectra. This pattern lacks any noticeable orientation dependence with a splitting of 4.3 MHz for SQA and 3.8 MHz for SQB. Shoulders on both sides of the sharp feature are resolved, and suggest the 14-17 MHz region may comprise two or three overlapping 13

C tensors.

Figure 6. Q-band 13C Davies ENDOR spectra of SQA (left) and SQB (right). Traces were taken at nine field positions spanning gX, gY, and gZ in steps of 0.5 mT. The experimental data is shown in black and is overlaid by the simulations in red. The simulations were only fit to the higher frequency half of the spectra (13-25 MHz). Experimental parameters: π/2-pulse length = 100 ns, time between first and second pulses τ = 500 ns, RF π-pulse length = 30 µs, microwave frequency = 34.176 GHz (SQA) and 34.114 GHz (SQB), temperature = 80 K.

18


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Q-band

13

C ENDOR simulations were initially attempted with 1C, 1C’, 2C, 2C’, 3C, 4C,

and 5C in Table 1 modeled as axial hyperfine tensors with A|| set collinear to gZ. Collinearity of A|| and gZ for the ring carbons is reasonably supported by DFT calculations on SQA and SQB (Figures S13 and S14). However, a satisfactory fit to the experimental ENDOR spectra could not be obtained under this model. The ENDOR spectra could only be simulated when (a) the pair of cross-ridges 1C and 1C’ were assigned to the same rhombic tensor (referred to as 1C-1C’ from here on), and (b) 2C and 2C’ were also assigned to a rhombic tensor 2C-2C’. For example, in the case of rhombic hyperfine tensor 1C-1C’, AX = A⊥ of 1C, AY = A⊥ of 1C’, and AZ = the average value of A|| from 1C and 1C’. AX, AY, and AZ for 2C-2C’ were assigned in an analogous fashion. Under this interpretation of the hyperfine tensors, the values of A|| in Table 1 for 1C and 1C’, and 2C and 2C’, should be equal. Differences in these extrapolated A|| values from the linear regressions in (ν1)2 vs (ν2)2 coordinates reflect the low signal-to-noise and poor resolution of some of these cross-ridges. Nevertheless, the SQA and SQB ENDOR spectra in the region 17-20 MHz were found to be well-fit to this model with two rhombic tensors (1C-1C’ and 2C-2C’) and three axial tensors (3C, 4C, and 5C). In the case of SQB, 3C and 4C were not resolved in the HYSCORE spectra, and were modeled as overlapping equivalent hyperfine tensors which significantly improved the fit of the simulations to the experimental ENDOR spectra. We then focused our attention on fitting the 14-17 MHz region of the ENDOR spectra. The intensive sharp peaks at 15.2 MHz for SQA and 14.9 MHz for SQB are best simulated with an essentially isotropic coupling |a| ≈ 4.3 and 3.8 MHz, respectively, with very low hyperfine anisotropy (6C). On the other hand, how simulations should be performed for the shoulders on either side of this peak is unclear, and no guidance can be obtained from Q-band HYSCORE where these peaks were undetected. Cross-ridges with similar splittings ~4-5 MHz were, 19



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Page 20 of 46

however, observed in the X-band HYSCORE spectra (asterisks in Figure 2). Linear regressions of these cross-ridges in (ν1)2 vs (ν2)2 coordinates identified

13

C couplings with a = 4.6±0.6 or -

5.2±0.6 MHz and T = 0.7±0.1 MHz for SQA, and a = 4.0±0.1 or -4.5±0.1 MHz and T = 0.5±0.1 MHz for SQB. When introduced into the ENDOR simulations, it was found that hyperfine tensors with a = 4.2 MHz and T = 0.7 MHz for SQA and a = 3.7 MHz and T = 0.6 MHz for SQB (7C) fit optimally with the experimental traces with Euler angle β set to 40°. While a fairly good fit could be obtained by modeling the 14-17 MHz region of the ENDOR spectra with just 6C and 7C, the shoulder around 15.5-16 MHz is not completely reproduced under this assumption, suggesting the presence of an additional tensor not yet accounted for. After a consideration of the available

13

C ring carbon hyperfine data in the

literature34, 44 and the DFT calculations (as expanded upon in greater detail in the Discussion), the 15.5-16 MHz peak was assigned to the C5 ring carbon. An additional tensor 8C was therefore included into the ENDOR simulations with A⊥ set to the 15.5-16 MHz shoulder, and the A|| component was taken from DFT calculations for C5 (8.0 MHz for SQA and 11.3 MHz for SQB). The ENDOR simulations were optimized by a constrained Nelder-Mead style leastsquares fitting routine45 allowing for full rhombic hyperfine tensors, where solutions not reproducing the experimentally observed peak locations were rejected. For the highly anisotropic tensors 1C-1C’, 2C-2C’, 3C, 4C, 5C, and 8C, the parallel components (AZ) were too weak in signal intensity in the ENDOR spectrum to be reliably simulated. Therefore, the AZ values were held fixed to the values found from linear regression analysis of the Q-band HYSCORE spectra in (ν1)2 vs (ν2)2 coordinates (Table 1) or the DFT calculations (for the tensor 8C assigned to C5). Therefore, only AX and AY for the anisotropic

13

C ring carbon couplings were optimized in the

ENDOR simulations. Simulated spectra were found to be very insensitive to the directions of the 20


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AX and AY axes within the gX/gY plane, so they were fixed collinear to the g-tensor axes (all Euler angles set to zero). For 6C and 7C, all principal values were optimized. The tensor 6C was far too isotropic for any meaningful optimization of the Euler angles, so they were all set to zero. For 7C, Euler angle β was set to 40° with all other Euler angles set to zero with no further optimization for both SQA and SQB. The results of the simulations are shown in red in Figure 6, with the parameters listed in Table 2.

Table 2. SQA and SQB 13C hyperfine simulation parameters (MHz) and comparison with DFT calculations in parentheses.a Positionb

Hyperfine Tensor

C1

1C-1C’

C2

3C

C3

5C

C4

2C-2C’

C5

8C

C6

4C

CH2

6C

CH

7C

SQA AX, AY, AZ -13.6±0.5 (-15.0) -16.0±1.0 (-18.5) 25.3c (13.3) -8.7±0.3 (-6.6) -8.6±0.4 (-5.9) 16.8c (20.9) -11.2±0.2 (-11.4) -10.8±0.3 (-11.2) (-9.6) -4.1c -8.4±0.6 (-7.7) -10.5±1.1 (-10.1) 35.7c (36.2) -5.3±0.7 (-8.7) -6.0±0.7 (-9.0) 8.0d (8.0) -8.2±0.3 (-7.2) -7.5±0.5 (-6.8) 14.8c (15.9) -4.4±0.3 (-4.5) -4.5±0.4 (-3.4) -4.3±0.3 (-4.5) 3.6±0.8 (5.1) 3.4±1.0 (5.2) 5.6±0.3 (6.6)

SQB

a, T, δ -1.4±0.5 (-6.7) 13.4±0.2 (10.0) 0.09±0.03 (0.17) -0.2±0.2 (2.8) 8.5±0.1 (9.1) 0.01±0.02 (0.04) -8.7±0.2 (-10.7) 2.3±0.1 (0.6) 0.08±0.05 (0.18) 5.6±0.5 (6.1) 15.1±0.3 (15.0) 0.07±0.03 (0.08) -1.1±0.5 (-3.2) 4.5±0.2 (5.6) 0.08±0.07 (0.03) -0.3±0.3 (0.6) 7.6±0.1 (7.6) 0.05±0.03 (0.03) -4.4±0.3 (-4.1) 0.0±0.2 (0.4) 0.6±0.5 (0.00) 4.2±0.7 (5.6) 0.7±0.4 (0.5) 0.1±1.0 (0.10)

AX, AY, AZ -11.7±0.7 (-13.0) -13.9±1.2 (-15.9) 30.1c (22.0) -9.0±0.3 (-8.0) -8.4±0.4 (-7.3) 10.9c (16.8) -10.1±0.3 (-8.8) -9.5±0.9 (-8.5) 6.0c (-1.0) -11.0±1.0 (-10.0) -13.1±1.5 (-12.4) 34.9c (31.8) -4.2±0.5 (-7.5) -4.6±0.7 (-7.9) 11.3d (11.3) -8.4±0.5 (-8.2) -8.2±0.5 (-7.8) 10.9c (11.6) -3.9±0.2 (-3.9) -3.8±0.3 (-3.0) -3.8±0.2 (-4.2) 3.2±0.7 (5.4) 3.0±0.9 (5.7) 4.9±0.4 (7.0)

a, T, δ 1.5±0.6 (-2.3) 14.3±0.3 (12.2) 0.08±0.03 (0.12) -2.2±0.2 (0.5) 6.5±0.1 (8.2) 0.05±0.03 (0.04) -4.5±0.4 (-6.1) 5.3±0.2 (2.6) 0.06±0.06 (0.06) 3.6±0.8 (3.1) 15.7±0.4 (14.3) 0.07±0.04 (0.08) 0.8±0.4 (-1.4) 5.2±0.2 (6.3) 0.04±0.06 (0.03) -1.9±0.3 (-1.5) 6.4±0.2 (6.5) 0.02±0.04 (0.03) -3.8±0.2 (-3.7) 0.0±0.1 (0.4) 1.0±0.5 (0.43) 3.7±0.6 (6.0) 0.6±0.4 (0.5) 0.2±0.8 (0.31)

a Principal values of the rhombic hyperfine tensor: AX = |a − T(1 + δ)|, AY = |a − T(1 − δ)|, and AZ = |a + 2T|, where δ is unitless and ranges from 0 to 1 corresponding to axial and rhombic tensors, respectively. The principal axes are assumed collinear with the g-tensor reference frame (AX, AY, and AZ point along gX, gY, and gZ, respectively) for the simulated hyperfine tensors. The DFT

21



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Page 22 of 46

principal values are also ordered under the same approximation. This model was reasonable for all carbons except CH, where the simulations required that Euler angle β be set to ~40° for a good fit. This is in good agreement with DFT calculations, where AZ is predicted to be skewed significantly by 39.7° and 50.1° from gZ for SQA and SQB, respectively. b C1 through C6 refer to the quinone ring carbons as numbered in Figure 1. CH2 and CH are the first and second, respectively, carbons of the isoprenoid tail. c AZ was fixed to the extrapolated value of A|| from squared-frequency linear regression analysis of the Q-band HYSCORE spectra. d AZ was fixed to the DFT calculated value of AZ for ring carbon atom C5.

Discussion Assignments of the

13

C Tensors to Specific Carbons. The ENDOR simulation

parameters for the eight detected

13

C nuclei are shown in Table 2. The assignments of these

hyperfine tensors to specific carbons can be made by comparison with DFT calculations and available 13C data on the RC semiquinones. Previously, SQA and SQB were 13C-labeled at the C1 or C4 positions, providing estimates of AX, AY, and AZ by simulations of the Q-band CW EPR spectra.34,

44, 46

The well-resolved hyperfine splitting at the gZ orientation allowed for a

determination of AZ for C1 and C4 as 22.7 and 35.0 MHz (SQA) and 27.7 and 32.2 MHz (SQB), respectively, all with approximate errors ±0.5 MHz. This provides an unambiguous assignment of the 1C-1C’ and 2C-2C’ cross-ridge pairs from the Q-band HYSCORE analysis as belonging to C1 and C4, respectively (Table 1). DFT calculations predict a hyperfine rhombicity for these two carbons that matches well with the ENDOR simulations (Table 2). Additionally, for both SQA and SQB, only 5C satisfies the unusual characteristics of the calculated C3 tensor with a strongly negative isotropic constant (a) and a moderate anisotropic coupling (T), and is therefore assigned to this carbon. The assignments of the 13C tensors to the remaining ring carbons (C2, C5, and C6) can be performed for SQA by considering available Q-band CW EPR 13C ring data and the DFT models. Previously, C5 and C6 in SQA were site-specifically

13

C-labeled and subjected to Q-band CW

EPR measurements,44 where the C6 AZ component of the hyperfine tensor was successfully 22


Page 23 of 46

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resolved and estimated to be 15.4±0.9 MHz. Only 4C of SQA has an AZ component within error of this value (14.8 MHz), and is therefore assigned to C6. For C5 of SQA, simulations of the EPR linewidth suggested that AZ is ~7 MHz or less.44 Since this value is incompatible with the AZ determined for 3C (16.8 MHz), 3C must instead be assigned to C2. SQA DFT calculations for C2 are in good agreement with 3C, supporting this assignment (Table 2). As a result, 8C is assigned to C5 by process of elimination. AZ could not be determined experimentally for 8C and was therefore set to the DFT calculated value of 8.0 MHz in the ENDOR simulations, in reasonable agreement with the previous studies.44 While the assignments of 3C, 4C, and 8C to C2, C6, and C5 (respectively) only apply to SQA, these arguments have been applied analogously to assign the resonances in the case of SQB. This is based on the assumption that the spin density distributions of SQA and SQB will exhibit similar trends in relative spin populations, as supported by the DFT models. An unambiguous assignment of the resonances will require site specific 13C labeling at the C2, C3, C5 and C6 positions.44, 47-49 While not detected in the Q-band HYSCORE spectra, ENDOR simulations revealed two 13

C couplings 6C and 7C having characteristics incompatible with that of a quinone ring carbon.

These strongly isotropic hyperfine tensors must therefore belong to the carbons of the isoprenoid tail. The first carbon of the isoprenoid tail (CH2) is calculated to have negative isotropic constants of -4.1 and -3.7 MHz for SQA and SQB, respectively. DFT has been found to reproduce with high reliability the 5’-methyl

13

C couplings in various SQ systems,25 so a similar level of

agreement is expected for the 6’-methylene carbon. Indeed, the calculated values agree very well with the experimental 6C isotropic constants of -4.3 MHz for SQA and -3.8 MHz for SQB, and can therefore be assigned to CH2. As a result, the remaining

13

C tensor 7C from ENDOR

simulations is assigned to the second carbon of the isoprenoid tail (CH). The isotropic constants 23



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Page 24 of 46

do not completely match the DFT calculations (especially for SQB), but similarly to the

13

C

methoxy couplings studied previously,25 the isotropic interaction with this nucleus is expected to be very sensitive to the orientation of the isoprenoid tail that may not be completely accurate in the DFT model. Nevertheless, the ENDOR simulation requirement for Euler angle β ~40° for this hyperfine tensor is in good agreement with the DFT result that AZ is significantly skewed by 39.7° and 50.1° from gZ for SQA and SQB, respectively. The assignments of all

13

C hyperfine

tensors obtained from the analysis of the spectroscopic data to the quinone ring and isoprenoid tail carbons is summarized in Table 2, with the principal values (AX, AY, AZ) listed alongside the corresponding isotropic (a), anisotropic (T), and rhombic (δ) coupling constants.

24


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Figure 7. Maps of the experimental s- (top) and pπ- (bottom) spin density distributions based on the past three decades of EPR research on SQA (left) and SQB (right).25,

34, 43

Positive and

negative spin density is represented as blue and red transparent spheres, respectively, where the areas indicate the relative spin populations. All spin densities shown here are listed in Table 3.

25



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Page 26 of 46

Maps of the SQA and SQB Spin Density Distributions. The past three decades of EPR research have elucidated nearly all of the major hyperfine interactions between the RC semiquinones and the nuclei that make up the protein, solvent, and quinone molecule itself. These include CW EPR studies of the 17O and 13C labeled quinone carbonyls and ring carbons,34, 44, 46, 50 13

C and 1H work on the quinone headgroup methoxy and methyl substituents,25, 34, 50-53 a

determination of the hydrogen bond networks by 1H ENDOR and HYSCORE,15, 32, 34, 35, 54-57 14,15

N studies of the histidine and peptide H-bond donors,35, 43, 58-62 and now finally a 13C pulsed

EPR investigation of the quinone ring and isoprenoid tail carbons. These studies have uncovered many high-resolution structural and functional properties of SQA and SQB that are extendable to other SQ systems. The isotropic and anisotropic components of the hyperfine tensor are directly related to the electron spin population in the s-orbital (Fermi contact) and the through-space magnetic coupling between the SQ spin and the nuclear spin (dipole-dipole interaction), respectively. In the case of the quinone ring carbons and carbonyl oxygens, the magnetic dipolar interaction is mainly due to unpaired electron spin density occupying the pπ-orbitals of the conjugated quinone ring system. The s- and pπ-spin populations for these atoms can therefore be estimated by dividing the isotropic (a) and anisotropic (T) constants, respectively, by their corresponding atomic hyperfine constants (a = 3777 MHz and T = 107.4 MHz for 13C, and a = -5263 MHz and T = -168.4 MHz for 17O).63 This procedure can also be performed for the 1H and 13C nuclei of the quinone substituents and

14

N hydrogen bond donors to extract the unpaired s-populations that

make up the spin density distribution. The maps of the unpaired s- and pπ-spin density distributions for SQA and SQB are visually represented in Figure 7. The hyperfine constants and calculated spin populations used to 26


Page 27 of 46

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generate Figure 7 are provided in Tables 3 and 4. Summation of all the spin densities collected thus far results in 87% for SQA and 89% for SQB. Not accounted for in the model are the 2-3% spin on the methoxy oxygens and the

Determination of the Complete Spin Density ... - ACS Publications

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