Two-Dimensional Pulsed EPR Resolves Hyperfine Coupling Strain in

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Two-Dimensional Pulsed EPR Resolves Hyperfine Coupling Strain in Nitrogen Hydrogen Bond Donors of Semiquinone Intermediates Sergei A. Dikanov, and Alexander T. Taguchi J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b02511 • Publication Date (Web): 26 Apr 2018 Downloaded from http://pubs.acs.org on April 27, 2018

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The Journal of Physical Chemistry

Two-Dimensional Pulsed EPR Resolves Hyperfine Coupling Strain in Nitrogen Hydrogen Bond Donors of Semiquinone Intermediates Sergei A. Dikanov,‡* Alexander T. Taguchi†,‡,@ ‡

Department of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States †

Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States

@

Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, United States

Corresponding Author *

E-mail: [email protected] (S.A.D.).

1 ACS Paragon Plus Environment

The Journal of Physical Chemistry 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

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ABSTRACT. Hydrogen bonding between SQ intermediates and sidechain or backbone nitrogens in protein quinone processing sites (Q-sites) is a common motif. Previous studies on SQs from multiple protein environments have reported specific features in the

15

N HYSCORE spectra not

reproducible by a theory based on fixed hyperfine parameters, and the source of these lineshape distortions remained unknown. In this work, using the spectra of the SQ in the Q-sites of wildtype and mutant D75H cytochrome bo3 ubiquinol oxidase from E. coli, we have explained the observed additional features as originating from a-strain of the isotropic hyperfine coupling. In 2D spectra the a-strain manifests as well-resolved lineshape distortions of the basic cross-ridges and accompanying lines of low intensity in the opposite quadrant that allow its direct analysis. We have shown that their appearance is regulated by the relative values of the strain width ∆a and parameter δ = |2a+T|-4ν15N. a-strain provides a direct measure of the structural dynamics and heterogeneity of the O⋅⋅⋅H⋅⋅⋅N bond in the SQ systems.


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INTRODUCTION Quinones are central components of most respiratory and photosynthetic electron-transfer chains.1,2 They mediate electron and proton transfer reactions in quinone binding sites (Q-sites) of proteins and shuttle redox equivalents between membrane enzymes. The diverse chemistry catalyzed by Q-sites proceeds through one or two-electron steps, and is often coupled to the net release or uptake of protons essential for completion of the redox reactions. The process of proton and electron conduction in Q-sites involves the binding of quinone and quinol as well as the reactive intermediate quinone species such as the semiquinone (SQ) radical (QH· or Q·-) and the deprotonated quinol (QH-). Limited resolution and ambiguities in the redox states of the quinones in available X-ray structures prevents a full description of specific interactions with the protein environment for Q-sites in different redox states Q/SQ/QH2.1-5 Spectroscopy and computations provide an alternative approach to probing the electronic structural properties of the quinones.6-9 The most direct way to characterize the intermediate SQ state is through EPR, exploiting its paramagnetism that eliminates ambiguities with respect to site occupancy due to diamagnetic species. High resolution EPR techniques such as Electron-Nuclear DOuble Resonance (ENDOR) and Electron Spin Echo Envelope Modulation (ESEEM) were used to explore the fine-tuning of the environment and electronic structure6-9 through the isotropic and anisotropic hyperfine interactions (hfi) and nuclear quadrupole interactions (nqi) with nearby magnetic nuclei. These magnetic interactions depend on the structure local to the SQ and its electronic state. All the SQs studied so far by ESEEM show hydrogen bond formation with one or more nitrogens.9 This kind of interaction is accompanied by delocalization of the unpaired spin density from the SQ onto the nitrogen donor manifesting in the appearance of an isotropic hyperfine coupling from spin density transferred onto the 2s orbital. During the last fifteen years, substantial progress has been achieved in the characterization of Q-sites in the SQ state using two-dimensional (2D) ESEEM, particularly its most popular 4-pulse version called HYSCORE.9 Uniform or selective

15

N (nuclear spin I = 1/2) protein labeling has been widely used in these

studies. The theoretical background of the cross-peak lineshapes in

15

N 2D ESEEM spectra,

which are free from complications of the nqi as in the case of an 14N nucleus with I = 1,10 allows for a simple, direct estimate of the isotropic and anisotropic constants of the hfi tensor. However, in our recent study of the SQ intermediate in Bacillus subtilis cytochrome aa3-600 menaquinol 3 ACS Paragon Plus Environment


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oxidase (cyt aa3-600) it was found that the HYSCORE spectrum of the

15

N hydrogen bond

donor, with isotropic a and anisotropic T components of the hfi tensor satisfying the specific condition δ = |2a+T|-4ν15N ≈ 0, showed well-pronounced distortions not describable by a single set of hyperfine parameters.11 The experimentally observed lineshapes were reproduced under the assumption of a span of 15N isotropic couplings with a distribution width of the order 10-15% from the average a-value, that is, “a-strain”, resulting from fluctuations in the protein environment. In this work we report the distortions of the cross-feature line-shapes under the influence of “a-strain” in the HYSCORE spectra of other semiquinones and analyze their appearance in the general case of an arbitrary deviation of the parameter δ from zero.

THEOPRETICAL BACKGROND ENDOR and ESEEM spectra show frequencies of transitions from nuclei interacting with the S = 1/2 electron spin of the SQ. A nucleus with I = 1/2 has two hyperfine frequencies, να and νβ, corresponding to the two states ms = ±1/2 of the electron spin in the applied magnetic field. HYSCORE is a technique which generates cross-features correlating the resonant frequencies να and νβ of a nuclear spin from opposite electron spin manifolds.12 HYSCORE spectra are sensitive to the relative signs of frequencies involved in the correlation13 and are usually represented by two quadrants, (++) and (+−), of the 2D Fourier transform. A nucleus with I = 1/2 may produce a pair of cross-peaks (να, νβ) and (νβ, να) in the (++) quadrant, and another pair (−να, νβ) and (να, −νβ) in the (+−) quadrant. The (|να|, |νβ|) cross-peak intensity is generally distributed between both quadrants of the HYSCORE spectrum for oriented systems (see the Supporting Info).14 In a powder-type 2D spectrum, να and νβ vary in a correlated manner over the entire interval between να(β)⊥ = |νI±(a‒T)/2| and να(β)|| = |νI±(a+2T)/2| forming a pair of cross-ridges described by the equation10:

ν α ( β ) = (Qα ( β )ν β2 (α ) + Gα ( β ) )

1/ 2

(1)

where

Qα ( β ) =

T + 2a − (+)4ν I T + 2a + (−)4ν I

Gα ( β ) =

(

+ (−)2ν I 4ν I2 − a 2 + 2T 2 − aT T + 2a + (−)4ν I

)

Qα(β) and Gα(β) are coefficients that are functions of a, T, and the Zeeman frequency νI = ν15N in this work. The ridge or contour lineshape forms a smooth arc extending between the points 4 ACS Paragon Plus Environment

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(να(β)⊥, νβ(α)⊥) and (να(β)||, νβ(α)||) located on the |να ± νβ| = 2νI lines and is confined to the area within these lines. However, for powder samples the phase interference between coherences from different orientations of the paramagnetic species becomes dominant that leads to essentially complete disappearance of the cross-ridges with Qα(β) > 0 for |2a+T| > 4νI oriented parallel to the main (positive) diagonal of the HYSCORE spectrum in the (++) quadrant. The opposite is also true in the (+‒) quadrant where cross-ridges with Qα(β) < 0 for |2a+T| < 4νI nearly perpendicular to the main (negative) diagonal of HYSCORE spectra are suppressed.14

RESULTS AND DISCUSSION Reported hfi tensors for 15N nitrogens involved in H-bonds with the SQ intermediates in different proteins show significant variations in the isotropic coupling (Table 1). In contrast, the Table 1. Reported isotropic and anisotropic hyperfine couplings and corresponding parameters 2a+T and δ for nitrogen donors hydrogen bonded with intermediate semiquinones in different proteins.

Quinone

Nitrogen Donor

a, MHz

T, MHz

2a+T, MHz

δ,a MHz

Ref.

QH cyt bo3

UQ8b

-0.8

15

UQ8

0.44g ~0.35 0.45g ~0.35 ~0.45

5.2

QH D75H cyt bo3

2.4 0.3 3.5 0.8 0.6 ~1-1.3

7.4

1.4

15

Q-site, Protein

QH cyt aa3-600

MQ7c

Nε-Arg71 Nε-His98 Nε-His75 Nε-His98 Nε-Arg71 Nε-Arg70

QH R70H cyt aa3-600 QA RCd

MQ7

N-His70

2.8

0.4

6.0

~0

11

UQ10 UQ10

3.2 3.6 1.9 0.6

6.8 7.6 4.3

0.8 1.6 -1.7

Qi cyt bc1e

UQ10

~1.1

0.4 0.4 0.49 0.2 ~0.2

17

QB RC

Nδ His-M219 Np Ala-M260 Nδ His-L190 Np Gly-L225 Nε-His217

QD NarGHIf

MQ8

Nδ His-66

~1.0

0.3

2.3

16

a

18 19

-3.7

20

δ = |2a+T|-4ν15N for ν15N = 1.5 MHz corresponding to the X-band experiment with external magnetic field ~350 mT; bUQ – ubiquinone; cMQ-menaquinone; dRC – bacterial reaction center from Rhodobacter sphaeroides; ecyt bc1 - ubiquinol: cyt c oxidoreductase from Rb. sphaeroides; fNarGHIE. coli nitrate reductase A, gobtained from averaging the two smaller principal components of the anisotropic hfi tensor



anisotropic components possess a stable value of the order of T15N ~0.4 MHz determined by the dipole-dipole interaction between the nitrogen and unpaired spin density localized on the pz orbital of the nearest carbonyl oxygen and on the p orbitals of the nitrogen.21 For illustration of the theoretical background discussed above, Figure S1 shows 15N HYSCORE spectra simulated with anisotropic component of the axial hfi tensor T = 0.4 MHz, isotropic coupling varying between 1.4 and 4.2 MHz, and ν15N =1.5 MHz. This range of isotropic coupling constants corresponds to |2a+T| changing in the interval from 3.2 to 8.8 MHz, and δ from -2.8 to 2.8 MHz. 15

N cross-features are located in the (++) quadrant for 3.2 < |2a+T| < 5.8 MHz and in the (+−)

quadrant for 6.2 < |2a+T| < 8.8 MHz only. They appear in both quadrants simultaneously or have

Figure 1. Partial presentation of the contour (A, B) 15N HYSCORE spectra of the 15N uniformly labeled WT (A) and D75H (B) cyt bo3. Full (A) and (B) spectra are provided in Figure S2. Red dashed lines are defined by |ν1 ± ν2| = 2ν15N. Experimental parameters: magnetic field 345.2 mT (A) and 346.1 mT (B), time between first and second pulses τ = 136 ns, microwave frequency 9.702 GHz (A) and 9.704 GHz. The region of the spectra with the cross-features located near ν2 = 0 line is shown.


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comparable intensity in the very narrow interval |2a+T| = 4ν15N = 6.0 ± 0.2 MHz. The crossridges in these cases are parallel to one of the axes or deviate very slightly from this direction. The special condition |2a+T| = 4ν15N defines νβ(α)⊥ = −νβ(α)∥ = ±(3T/4), where all orientations contribute to the same frequency |3T/4|, resulting in a singularity for the nuclear transition in one manifold. In 2D spectra, the theoretically predicted lineshape becomes a straight-line segment parallel to the να(β) axis in the interval from να(β)⊥ = |a−T/4| to να(β)|| = |a+5T/4| at νβ(α) = |3T/4|.10,22 Most hfi tensors collected in Table 1 were initially obtained with high accuracy from a fit of the

15

2

2

N HYSCORE spectra in the squared frequency (ν1 vs.ν 2 ) representation using Eq. 1,10

and were further analyzed by numerical simulations simultaneously modeling the HYSCORE and/or

15

14

N

N ENDOR spectra. These simulations reproduce the location and total

length of the cross-features. However, in many cases distortions and additional minor features were visible in the opposite quadrant of the HYSCORE spectra. These features were not described by the simulations based on a single set of hfi couplings and their appearance remains unexplained. These additional features are illustrated by the 15N HYSCORE spectra of the SQ in wildtype and D75H mutant of cytochrome bo3 ubiquinol oxidase from E. coli (cyt bo3) in Figure 1. The isotropic couplings reported previously for nitrogen donors Nε-Arg71 (WT) and Nε-His75

Figure 2. Ideal contour lineshapes of cross-ridges from 15N with a = 2.4 MHz (1A) and 3.5 MHz (1B), T = 0.4 MHz, ν15N = 1.5 MHz calculated using Eq. 1. Red lines are defined by |να ± νβ| = 2ν15N. 7 ACS Paragon Plus Environment


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(D75H) are 2.4 (NA) and 3.5 (NB) MHz, and the corresponding parameters of δ are -0.8 and 1.4 MHz, respectively. The ideal theoretically predicted cross-ridge lineshapes are arcs 1A and 1B extending between two points on the |να ± νβ| = 2ν15N lines as stated above (Figure 2). For NB both (να⊥, νβ⊥) and (να||, νβ||) are located along the same να + νβ = 2ν15N line parallel to the diagonal in the (+‒) quadrant. In contrast, for NA only (να⊥, νβ⊥) is on the extension of this line in the (++) quadrant but the (να||, νβ||) point belongs to the να - νβ = 2ν15N line parallel to the diagonal of this quadrant. HYSCORE spectra calculated for a span of isotropic couplings including 2.4 MHz (for spectrum A) and 3.5 MHz (for spectrum B) are provided in Figure S1. They show the same locations of the cross-ridges as in Figure 2 but with shorter length because intensity at (να(β)⊥, νβ(α)⊥) and (να(β)||, νβ(α)||) is equal to zero and is significantly suppressed at orientations around the principal directions.14 Comparison of the experimental and calculated spectra (Figures 1, 2, and S1) indicates that the major features 1A,B possess curvatures reversed relative to the arc shape in the calculated spectra. In addition, short cross-ridges of low intensity (1’A,B) parallel to the coordinate axes are observed in opposite quadrants of both spectra. The X-band 15N HYSCORE spectrum of the SQ in cyt aa3-600 satisfying the relationship δ ≈ 0 mentioned above is shown in Figure 3.11 The cross-ridges from the

15

N H-bond donor in

this spectrum contain a straight section parallel to the ν1 or ν2 axis to good accuracy as well as an additional extension along the red dashed line described by ν1 + ν2 = 2ν15N (Figures 3 and S3). Again, the exhibited “boomerang-type” curvature is inconsistent with the straight segments (Figure S1) theoretically predicted for a single hfi tensor with δ = 0. The experimentally observed line shapes were reproduced under the assumption of

15

N isotropic hfi coupling “a-

strain” of the order σ15N = 0.35 MHz with a Gaussian distribution model (Figure S4).11 It is reasonable to suggest that the a-strain would influence the cross-ridge shape at any value of the hfi coupling. However, clearly resolved effects from the a-strain in the form of the “boomerang” lineshape and low intensity lines in the opposite quadrant of the HYSCORE spectrum are only expected to appear when part of the strain broadened cross-ridge approaches or satisfies the condition (2(a±∆a)+T) ~ 4νI. The intensity and shape of the cross-features in the opposite quadrant will depend on the strain distribution function and the ∆a and δ values.


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(+‒)

(++)

Figure 3. Partial presentation of the contour 15N HYSCORE spectrum of the SQ in 15N uniformly labeled R70H cyt aa3-600. The full spectrum is shown in Figure S2. Red dashed lines are defined by |ν1 ± ν2| = 2ν15N. Experimental parameters: magnetic field 343.25 mT, time between first and second pulses τ = 136 ns, microwave frequency 9.625 GHz, temperature 60 K. The region of the spectra with the cross-features located near the ν2 = 0 line is shown.

We modeled the appearance of the strain-produced effects using hfi tensors for the nitrogens NA (a = 2.4 MHz, δ = ‒0.8 MHz) and NB (a = 3.4 MHz, δ = 1.2 MHz) with a triangle distribution function for the isotropic coupling f(∆a) = (∆a0)-1(1 ‒ |∆a/∆a0|) (∆a is the deviation in MHz units from the central isotropic constant a, and varies between –∆a0 and ∆a0) (Figures S5 and S6). In the triangle distribution, ∆a0 is the distance between the selected central frequency of maximum intensity (a) and the frequency where of the distribution function reaches zero. This function with a limited distribution interval allows us to establish exact conditions for the appearance of “strain” produced cross-features. Changes in a are targeted in this study, because we previously found a (but not T) to be very sensitive to the histidine Nδ-SQ H-bond length, resulting in reported isotropic couplings that vary by up to a factor of ~3 (Table 1).17 The anisotropic coupling with nitrogens is always at least several times smaller and variations of this coupling are only of a few hundred kHz and would not explain the observed spectral changes. The anisotropic contribution has been ignored at this stage of analysis. Spectra simulated with different values of ∆a0 have allowed us to characterize the following peculiarities of the influence of a-strain on HYSCORE spectra applicable for both nitrogens. The strain has almost no influence on the cross-ridge shape for ∆a0 < |δ/4| (Figure S6) 9 ACS Paragon Plus Environment


as in the ideal limit shown in Figures 2 and S1. Progressive deviations of the cross-ridges from their initial shape accompanied by a change in curvature and formation of a tail parallel to the nearest ν1 or ν2 axis (at the (να||,νβ||) edge for NA or (να⊥, νβ⊥) edge for NB, see Table S1) take place in the interval |δ/4| < ∆a0 < |δ/2|. In this interval the condition (2(a ± ∆a)+T) ~ 4ν15N or δ ± 2∆a ~ 0 (depending on the sign of δ) is approached that would lead to the appearance of subsections of the cross-ridges with a heterogeneous mixture of Qα ~ (δ ‒(+) 2∆a)/8ν15N and Qβ = 1/Qα (Figure S7) and would explain the observed behavior in lineshape changes. The condition δ ± 2∆a = 0 becomes satisfied at the distribution width ∆a0 = |δ|/2. The tail in the form of a straight segment with ν1(2) = |3T/4| appears in the simulated spectra at ∆a0 ~ |3δ/4| when part of the broadened cross-ridge possesses sufficient intensity for its observation. Simultaneously, low intensity features parallel to the coordinate axes begin to appear in the opposite quadrant and reach an intensity maximum at ∆a0 ~ |δ|. ∆a ~ δ/2 is necessary to observe these weak lines, corresponding to the half-height intensity of the triangle distribution. A further increase of ∆a0 leads to the extension of this new ridge along the ν1 + ν2 = 2ν15N line. Simultaneously, the edge of cross-ridges remote from the ν1(2) = |3T/4| lines stretch along the ν1 + ν2 = 2ν15N lines producing the illusion of an increased anisotropic hfi (Figure S6). The specific changes discussed above in the spectra for the triangle distribution are similar to those calculated for spectra with a more realistic Gaussian distribution f(∆a) = [σ(2π)1/2]-1exp[-(∆a)2/2σ2] (σ is the standard deviation) but at lower σ/|δ| ratios. For instance, the initial changes in shape of the ridge are observed at σ > 0.15|δ|. The appearance of cross-ridges in the opposite quadrant appear at σ > |δ/2|, and are well observed at σ ~ |3δ/4|. Again, the condition |δ ± 2∆a| ~ 0 suggests that part of the broadened line with |δ| ~ σ contributes to the appearance of the straight segment and new weak line in the opposite quadrant. The half-height intensity in the Gaussian distribution corresponds to 1.18σ, and thus the appearance of ridges in the opposite quadrant takes place at a larger value of σ for the Gaussian broadening than for the triangle distribution half-height parameter. In order to compare ∆a0 from the triangle distribution function with the standard deviation of the Gaussian distribution (σ), one can assume approximate correspondence between ∆a0/2 and σ. However, generally, the distortive influence of the a-strain on the HYSCORE spectra will depend on the strain distribution function, its width, and δ value.


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Figure 4. Calculated HYSCORE spectra for nitrogens NA and NB showing the transformation of the cross-ridge shape as a function of the strain width parameter σ for a Gaussian distribution f(∆a) = [σ(2π)1/2]-1exp[-(∆a)2/2σ2] model of the a-strain. Spectra were calculated by summing HYSCORE simulations performed at 21 evenly spaced points along a Gaussian distribution between –2σ to +2σ. The experimental spectra provide direct quantitative information about the strain of the hfi coupling reflecting geometric fluctuations in the corresponding structures.

15

N HYSCORE

simulations of the SQs in WT and D75H cyt bo3 with a Gaussian distribution a-strain model are 11 ACS Paragon Plus Environment


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shown in Figure 5. The best fits of the major and minor peaks for WT cyt bo3 are obtained with the same parameters as previously reported with an axial hfi tensor a = 2.4 MHz, T = 0.4 MHz, and standard deviation σ = 0.3 MHz. For the D75H mutant of cyt bo3, the optimal parameters of the tensor slightly deviate from those previously reported as a = 3.3 MHz and T = 0.45 MHz, with σ = 0.3 MHz. For both nitrogens, the errors were found to be ±0.15 MHz for a and σ, and ±0.1 MHz for T.

Figure 5. Comparison of the experimental 15N HYSCORE spectra from Figure 1 with the spectra calculated with a Gaussian distribution of the isotropic hyperfine coupling. The best fit of the spectra was obtained with a = 2.4 MHz, T = 0.4 MHz, σ = 0.3 MHz for WT cyt bo3 (left) and a = 3.3 MHz, T = 0.45 MHz, σ = 0.35 MHz for D75H cyt bo3 (right). Spectra were calculated by summing HYSCORE simulations performed at 9 evenly spaced points along a Gaussian distribution between –2σ to +2σ. Full spectra are shown in Figure S8. The simulation parameters obtained define the relations σ = 0.33|δ| (WT) and σ = 0.38|δ| (D75H) that are smaller than the formulated condition σ > |δ|/2 expected for the appearance of the weak lines in case of the triangle and Gaussian distributions. These values of σ are similar to the σ = 0.35 MHz reported previously for the SQ in cyt aa3-600 with the

15

N spectrum

characterized by δ ~ 0.11 The similar values of σ across these different SQ systems likely reflects a similar mechanism of structural heterogeneity in the interaction of the SQ with the protein environment. The optimization of σ in the simulations is based more on matching the “boomerang” lineshape of the cross-ridges than the weak lineshape appearing in the opposite


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quadrant. On the other hand, for simulations of spectra with δ very near to 0 MHz, the boomerang lineshape must be reproduced in both quadrants. One can suggest that fluctuations of the SQ in the protein environment, producing proton structural deviations in the O⋅⋅⋅H⋅⋅⋅N bond, are responsible for the well-resolved strain effects. Model DFT simulations of the a(15N) dependence on O⋅⋅⋅H bond length for N donors in the QA site show that variations of the order ~0.3 MHz correspond to a distance change of ~0.1 Å.23 This estimate suggests that fluctuations in the O⋅⋅⋅H distance of the order ~0.1 Å can be responsible for the a-strain distortions in the 15N HYSCORE spectra of nitrogen H-bond donors. Although in this work we have focused on spectra with low hfi anisotropy, our simulations have shown that similar a-strain effects will be observed at larger values of T up to 1 MHz if the condition |2a+T| ~ 4νI is satisfied. Potentially one can expect the influence of a-strain effects from any I = 1/2 spin; typical of them in protein studies are 1H,

13

C,

15

N,

19

F, and

31

P.

Considering the properties of powder type X-band 2D spectra, the strain effects are most probable to be observed for

13

C and

31

P in addition to

15

N, because it requires nuclei with

moderate isotropic couplings of 5-10 MHz to satisfy the condition |2a+T| ~ 4νI (Table 2). However, by employing the multifrequency approach one can expand the range of hfi couplings suitable for observation of the a-strain effects to lower and/or higher values. Table 2. 4ν νI values for different I = 1/2 nuclei in X-band.a 1 13 15 19 31 Nucleus H C N F P 59.6 15.0 6.04 56.11 24.15 4νI, MHz a

for magnetic field 350 mT.

The strain produced features of the contour lineshapes resolved in HYSCORE are also predictable in spectra from any 2D pulse sequence correlating να and νβ. This is because Eq. 1 for the cross-ridge lineshape has a general characteristic depending exclusively on the hyperfine couplings. The particular type of sequence will only influence the intensity profile along the cross-ridges. g- and A–strains were previously analyzed in EPR spectra of metallocomplexes and clusters.24-26 However, in 1D spectra, several factors usually contribute to the linewidth and significant efforts are needed to quantify the separate contributions from g- and A-strain. In 2D spectra a-strain is purely seen as line-shape distortions and new lines of low intensity wellseparated from major peaks that allow its direct analysis.



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The analysis of the boomerang lineshape and its implications on molecular structure discussed here provide new insight into the structural dynamics in other spin systems where astrain effects have (or have not) been observed in HYSCORE spectra with the appropriate deviation from δ ≈ 0. First, a-strain in the spectra of the SQs from 15N H-bond donor(s) appears to be a general phenomenon extending beyond the cyt aa3-600 and cyt bo3 systems. These spin systems were studied here as a convenient example for the analysis of the a-strain phenomenon from single

15

N with arbitrary hyperfine parameters. The

15

N HYSCORE spectra for the SQ of

the QA site of the photosynthetic reaction center clearly display characteristics of a-strain not captured by our previous simulations which did not account for a-strain at that time.17 However, more complex analysis is required for this site where two H-bond N donors possessing very similar hyperfine couplings contribute to the spectra that would interweave the individual astrain features. In addition to SQs in proteins, a-strain effects can also be recognized in

31

P

HYSCORE spectra of vanadium complexes with phosphates.27-30 Despite the observation of astrain effects in HYSCORE spectra in previous works, these influences were ignored during the quantitative analyses and were never fully explained. On the other hand, the hyperfine interaction of the SQA spin with its

13

C(3) ring carbon

exactly satisfying δ ≈ 0 showed no observable a-strain effects in the 13C HYSCORE spectrum.31 Similarly, the pyrrole nitrogens in a model NO-coordinated iron porphyrin showed no resolvable a-strain contributions despite satisfying δ ≈ 0.32 This consideration allows us to suggest that 2D spectra would not exhibit the influence of a-strain in systems with structural rigidity of the paramagnetic molecule, whereas a-strain would be observed when the electron spin is distributed through a more flexible metal coordination or hydrogen bond to the interacting nucleus.

ASSOCIATED CONTENT Supporting Information Intensity of cross-peaks in HYSCORE spectra. Figure S1, simulated 15N HYSCORE spectra with varying isotropic coupling and constant anisotropic coupling T = 0.4 MHz (15N), 4ν15N = 6.0 MHz. Figure S2, 15N HYSCORE spectra of the SQ in 15N uniformly labeled WT and D75H cyt bo3. Figure S3, 15N HYSCORE spectrum of the SQ in 15N uniformly labeled R70H cyt aa3-600. Figure S4, comparison of the experimental X-band 15N HYSCORE spectrum of SQ in cyt aa3600 with simulations using different values of the standard deviation σ for the Gaussian distribution. Figures S5, definition of the triangle distribution function. Figure S6, simulated 15N HYSCORE spectra with triangle distribution models of the a-strain with ∆a0 varying from 0.0 to 14 ACS Paragon Plus Environment

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2.0 MHz. Figure S7, dependence of Qα and Qβ as a function of ∆a for NA and NB. Figure S8, comparison of the experimental 15N HYSCORE spectra from Figure 1 (full presentation) with the spectra calculated with a Gaussian distribution of the isotropic hyperfine coupling. Table S1, components of the hyperfine tensor and corresponding nuclear frequencies for NA and NB. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *S.A.D.: email, [email protected]; phone, (217) 300-2209. ORCID Sergei A. Dikanov: 0000-0003-2610-6439 Alexander T. Taguchi: 0000-0002-5940-5948 Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

Acknowledgements This work was supported by Grant DE-FG02-08ER15960 (S.A.D.) from the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Sciences, U.S. Department of Energy, and NCRR/NIH Grants S10-RR15878 and S10-RR025438 for pulsed EPR instrumentation. A.T.T. gratefully acknowledges support as an NIH trainee of the Molecular Biophysics Training Program (5T32-GM008276). The authors thank Nikki Erika Riser for designing the Table of Contents (TOC) figure. References. 1. Quinones and quinone enzymes, Part A, Methods in Enzymology, 2004; Vol. 378. 2. Quinones and quinone enzymes, Part B, Methods in Enzymology, 2004; Vol. 382. 3. Ficher, N.; Rich, P. A Motif for Quinone Binding Sites in Respiratory and Photosynthetic systems. J. Mol. Biol. 2000, 296, 1153-1162. 4. Mitchell, P. Possible Molecular Mechanisms of the Proton Motive Function of Cytochrome Systems. J. Theor. Biol. 1976, 62, 327-367. 5. Functions of quinones in energy conserving systems; Trumpower, B. L., Ed.; Academic Press, 1982. 6. Lubitz, W.; Feher, G. EPR of Quinone Radicals in Photosynthetic Reaction Centers. Appl. Magn. Reson. 1999, 17, 1-49. 7. Lubitz, W. EPR in Photosynthesis. Electron Paramagn. Reson. 2004, 19, 174-242.



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Two-Dimensional Pulsed EPR Resolves Hyperfine Coupling Strain in

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