Regulation of the Primary Quinone Binding Conformation by the H

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Letter

Regulation of the Primary Quinone Binding Conformation by the H Subunit in Reaction Centers from Rhodobacter sphaeroides. Chang Sun, Alexander T. Taguchi, Nathan Beal, Patrick J. O'Malley, Sergei A. Dikanov, and Colin A. Wraight J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.5b01851 • Publication Date (Web): 30 Oct 2015 Downloaded from http://pubs.acs.org on November 1, 2015

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The Journal of Physical Chemistry Letters

Regulation of the Primary Quinone Binding Conformation by the H Subunit in Reaction Centers from Rhodobacter sphaeroides. Chang Sun,§ Alexander T. Taguchi,†,‡,@ Nathan J. Beal,& Patrick J. O’Malley,&* Sergei A. Dikanov,‡* Colin A. Wraight†,§,#

§

Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States †

Center for Biophysics and Computational Biology, University of Illinois at UrbanaChampaign, Urbana, Illinois 61801, United States &

‡

School of Chemistry, University of Manchester, Manchester M13 9PL, U.K.

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

#

Deceased on July 10, 2014

Corresponding Author *

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

*

E-mail: [email protected] (P.J.O.).

Present Address @ Department of Biochemistry and Molecular Biology, Nippon Medical School, Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan

1 ACS Paragon Plus Environment


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ABSTRACT. Unlike photosystem II (PSII) in higher plants, bacterial photosynthetic reaction centers (bRCs) from Proteobacteria have an additional peripheral membrane subunit “H”. The H subunit is necessary for photosynthetic growth, but can be removed chemically in vitro. The remaining LM dimer retains its activity to perform light-induced charge separation. Here we investigate the influence of the H subunit on interactions between the primary semiquinone and the protein matrix, using a combination of site-specific isotope labelling, pulsed EPR, and DFT calculations. The data reveal substantially weaker binding interactions between the primary semiquinone and the LM dimer than observed for the intact bRC; the amount of electron spin transferred to the nitrogen hydrogen bond donors is significantly reduced, the methoxy groups are more free to rotate, and the spectra indicate a heterogeneous mixture of bound semiquinone states. These results are consistent with a loosening of the primary quinone binding pocket in the absence of the H subunit.

TOC GRAPHICS

KEYWORDS. ubisemiquinone, pulsed EPR, hyperfine coupling, isotope labelling, sitedirected mutagenesis, DFT


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The bacterial photosynthetic reaction center (bRC) from Rb. sphaeroides has served as an exemplary model system in the field of bioenergetics, and has provided a great deal of insight into the inner workings of biological electron transfer (ET) and quinone redox chemistry.1 It has also found recent application as a tool for interfacial electrochemistry, chemical sensing, and energy conversion.2–4 This protein complex consists of two transmembrane subunits L and M where all of the photochemistry takes place (LM dimer), and a peripheral membrane subunit H (Figure 1). The H subunit has a single transmembrane helix and a large globular domain which caps the QA and QB quinone binding sites. The H subunit is asymmetrically positioned mostly over the QA site, but also provides key residues for proton delivery to the QB site.1 The essential role of the H subunit in regulating bRC assembly in Rb. sphaeroides and related species is also well-established.5–8 However, it is only conserved in photosynthetic proteobacteria and not in other type II bRCs (filamentous anoxygenic phototrophs, cyanobacteria, and plants). The H subunit can be removed in vitro by treatment with detergents or chaotropic agents, which has allowed for significant insight to be gained into the biochemical properties of the LM dimer.9–14 The LM dimer is structurally similar to PSII in plants and cyanobacteria, although it lacks the oxygen evolving complex in the M subunit (homologous to the D2 subunit in PSII). Functionally, the LM dimer retains the ability to form and stabilize the anionic QA- semiquinone (SQA) like the bRC, but is essentially incapable of the subsequent inter-quinone ET to QB that forms QB- (SQB).9,10 The original work of Debus et al.7 suggests that ubiquinone is still bound to the QB site, albeit more weakly, with an apparent SQA to QB ET rate almost 1000-fold slower than in the intact bRC.9 If this is correct, it may indicate that in the LM dimer the redox potential difference between the quinones is unsuitable for ET, which can be caused by changes in the local electrostatics in one or both quinone sites,15 or a failure of the methoxy groups to adopt the orientations necessary to bring the redox potential



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difference into the functional range.16–18 Redox titrations (Figure S1 in the SI) show the Em of QA in the LM dimer to be only slightly perturbed relative to the intact bRC. A similar redox titration for QB, however, has yet to be successfully performed, preventing a determination of the LM dimer quinone redox potential difference.

Figure 1. The reaction center (RC) complex from Rhodobacter sphaeroides comprises three subunits, a heterodimer of similar, but non-identical L (tan) and M (green) subunits, and subunit H (red) that caps LM on the cytosolic side of the membrane. The special pair of bacteriochlorophyll (P), bacteriochlorophyll (BA and BB), bacteriopheophytin (HA and HB), and the quinones (QA and QB) are shown in purple. The arrows indicate the initial charge separation (arrow from P to QA) and the subsequent interquinone ET (arrow from QA to QB).

Alternatively, the slower inter-quinone ET may reflect a decrease in the intrinsic kinetics of the ET rate. This would suggest a ~5 Å increase in distance between the quinones,19 which is unlikely if the quinone binding sites of the LM dimer still resemble that of the bRC. However, there is no structural information available so far for the LM dimer, regardless of the redox states of the quinones. Here, we report the first pulsed EPR study on the primary semiquinone in the LM dimer (SQLM).


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SQLM has been successfully generated by either chemical (dithionite) or photochemical (532 nm laser excitation) reduction. Because the chemical reduction method is not expected to generate SQB, we assign this signal to a SQ of the QA site. Furthermore, we have prepared the L223 serine to tryptophan mutant to test this. This mutation completely knocks out the binding of QB to the intact bRC. LM dimer preparations from this mutant give the same SQLM signal as indicated by the EPR and ESEEM spectra (Figures S2 and S3), confirming its identity as a QA site semiquinone (SQA only refers to the QA semiquinone in the WT bRC hereafter). Q-band continuous-wave EPR spectra of SQLM in these samples coincide with that observed for SQA (Figure S4), where the g-tensor has principal values 2.0065, 2.0053 and 2.0021.20 Despite the similarities in the CW EPR spectra, 1D and 2D ESEEM characterizations have revealed significant differences between the SQLM and the SQA. Figure 2a shows stacked plots of the three-pulse 14N ESEEM spectra of SQLM. The spectra possess a shape typical for a single 14N near cancellation conditions (ν14N ~ |a(14N)|/2) in one electron spin manifold21,22 (see also the SI). This allows for an immediate assignment of the narrow peaks at 0.55, 0.89, and 1.44 (±0.03) MHz to the three pure nuclear quadrupole interaction (nqi) frequencies ν0, ν, and ν+ (Eq. S1) within this manifold. There is also a much weaker and broader line at ~4.4 MHz, which is suggestive of the νdq+ transition (Eq. S3) from the opposite manifold. This assignment is confirmed by the

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N HYSCORE spectrum (Figures 2b and S2). The

spectrum exhibits cross-features 1-3 correlating ν0, ν-, and ν+ with νdq+, thus indicating that they belong to different electron spin manifolds.23 This correlation spectrum also allows for a more precise determination of the value of νdq+ = 4.1 MHz from the maximum intensity of the (νdq+, ν+) cross-peak 1. The frequencies ν0, ν-, ν+ and νdq+, along with the

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N Zeeman

frequency (ν14N = 1.06 MHz) can be used to estimate the quadrupole coupling constant (qcc) K = e2qQ/4h = 0.39 MHz, the asymmetry parameter η = 0.71, and the hyperfine interaction 5 ACS Paragon Plus Environment


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Figure 2. (a) Stacked plot of three-pulse ESEEM spectra of SQLM. The time-domain envelopes were recorded as a function of both T, the time between the second and third pulses, and τ, time between the first and second pulses. Spectra were obtained from modulus Fourier Transforms (FT) along the time T. The time τ was 100 ns for the back trace and was increased in steps of 16 ns in successive traces. (b) Contour representation of the 14N HYSCORE spectrum of SQLM. The time τ, the interval between the first and the second microwave pulses, was 136 ns. The spectrum was obtained by FT of the two-dimensional time domain patterns containing 256 x 256 points with a 20 ns step in t1 and t2, which are the intervals between the second and the third microwave pulses,


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and the third and the fourth microwave pulses, respectively. A 3D-type stacked presentation of this spectrum is shown in Figure S2. The microwave frequency was 9.635 GHz, the magnetic field was set to 343.5 mT. (c) Contour representation of the

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N HYSCORE spectrum of SQLM. The time τ was 136 ns. The microwave

frequency was 9.622 GHz, the magnetic field was 343.2 mT, and the temperature was 90 K. The spectrum was obtained by FT of the two-dimensional time domain patterns containing 256 x 256 points with a 32 ns step in t1 and t2.

(hfi) coupling a(14N) = 1.7 MHz for the 14N nucleus (Eqs. S1, S3, and S4).21-23 It is well established that the SQA is hydrogen-bonded to Nδ of His-M219 (which is also a ligand of a high spin nonheme Fe2+) and the peptide nitrogen (Np) of Ala-M260.24–26 The qcc of the nitrogen in the ESEEM spectra of SQLM is typical for Nδ of a histidine, indicating that SQLM retains the H-bond with His-M219. However, the SQLM spectra do not show the wellpronounced peptide nitrogen lines seen for SQA.25,26 An interaction with a peptide nitrogen (Np) is suggested by the presence of the low intensity broad cross-features 4 distributed within the area (4.0-4.7, 2.5-3.0) MHz in the 14N HYSCORE spectrum. Assuming the larger frequency corresponds to νdq+, we estimate the hfi coupling of Np is 1.28 ± 0.45 MHz from Eq. S4, when the typical values of K = 0.74 MHz and η ~ 0.5 reported for Np hydrogen bonded with SQA and SQB are used.26,27 The above assignments of the

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N peaks are also supported by the

15

N HYSCORE

spectrum of the SQLM. The (++) quadrant of this spectrum (Figure 2c) contains cross-features 1N and a sharp peak Nwc at (ν15N, ν15N). The cross-features 1N possess an asymmetric shape with an intensity maximum at (2.7, 0.4) MHz and long shoulders extending towards the diagonal. The first-order estimate of the hfi coupling based on the peak maximum is 2.3 MHz (1.6 MHz when scaled to 14

14

N). This value is consistent with the coupling obtained from the

N HYSCORE spectrum for the nitrogen assigned to Nδ of His-M219. The smallest splitting

between the low intensity shoulders of cross-feature 1N is ~1 MHz (~0.7 MHz for

14

N), in



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agreement with the smallest hfi coupling estimated for Np of Ala-M260 above. This suggests that the Np contributes to the extended low intensity part of the cross-features 1N in the

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N

HYSCORE spectrum. The sharp diagonal peak Nwc results from weakly coupled nitrogens around the SQLM in the protein environment.

Figure 3. Contour presentation of the 1H HYSCORE spectra of SQLM in protonated (a) and deuterated (b) solvent. The time τ was 136 ns. Spectra were obtained by Fourier Transforms of the two-dimensional time domain patterns containing 256 x 256 points with a 20 ns step in t1 and t2. The microwave frequency was 9.622 GHz, the magnetic field was 343.2 mT, and the temperature was 90 K.

Hydrogen bonds which stabilize SQLM were also examined by 1H HYSCORE (Figure 3). H-bonded protons typically possess anisotropic hfi couplings exceeding the couplings found for ubiquinone ring substituent protons (2,3-methoxy, 5-methyl and 6-methylene).28 As a result, protons from H-bonds produce extended cross-ridges shifted substantially from the dashed anti-diagonal line passing through (ν1H, ν1H) (Figure 3a). Two pairs of exchangeable shifted ridges (1H and 1’H) are observed in the 1H HYSCORE spectrum (Figure 3a) of SQLM.


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Similar patterns show up in the HYSCORE spectrum (Figure S5) with τ = 200 ns. Linear extrapolation of 1H in ν12 vs. ν22 coordinates coincides well with the 1’H segments located on the opposite side of the diagonal (Figure S6), suggesting that these features belong to the extended ridge from the same proton.28 This analysis provides the value TH = 4.4 MHz for the axial anisotropic hfi tensor (-TH, -TH, 2TH) (Figure S6) that is supported by the observation of only one sum combination peak in the four-pulse ESEEM spectrum where the peak shift from 2νH corresponds to a nearly identical TH ~ 4.5 MHz (Figure S7).

Figure 4. Contour presentation of the 13C HYSCORE spectrum of SQLM in the LM dimer with the ubiquinone head group 13CH3 and 13CH3O labelled. The time τ used was 136 ns. The microwave frequency was 9.642 GHz and the center of magnetic field was 343.8 mT, and temperature was 90 K. The spectrum was obtained by Fourier Transforms of the two-dimensional time domain patterns containing 256 x 256 points with a 20 ns step in t1 and t2. A stacked presentation of this spectrum is shown in Figure S8.

The

13

C HYSCORE spectrum of SQLM in the LM dimer with the ubiquinone head group

13

CH3 and 13CH3O labelled is dominated by broad cross-peaks with a maximum at (5.2, 2.2)



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MHz. This spectrum is more similar to that of the anionic SQ in alcohol solvent29 than that of SQA or SQB in the bRC, where three narrow cross-peaks with different couplings were resolved.30 Presumably, contributions from all three 13C labelled carbons are lumped together under the same peak. This suggests that the methoxy groups in the ubisemiquinone are less constricted in the LM dimer than in the bRC. They rotate more freely and take on a variety of conformations as they would in water or alcohol solvents. However, the cross-peak maxima are still significantly different in the LM dimer compared to the situation of organic solvents where hydrogen bonding to the carbonyl oxygens is symmetric.

Table 1. Comparison of select hfi and nqi coupling constants for SQA, SQB and SQLM. a (14N), MHz K = e2qQ/4h, MHz TH, MHz a(13C), MHz η 2.3a 0.38a 0.97a 5.2a 1.3 (2-CH3O) SQA b b b b ~0 (3-CH3O) 2.6 0.74 0.59 4.5-4.6 c c c c 1.4 0.38-0.39 0.69 4.5-4.7 4.7 (2-CH3O) SQB 1.5 (3-CH3O) 0.4-0.5d 0.74d 0.45d 3.2d SQLM ~1.7 0.39 0.71 4.4 ~3 a,b SQA: aNδ His-M219, bNp Ala-M260 from ref.26; c,dSQB: cNδ His-L190, dNp Gly-L225, from ref.27.

Previously, the hydrogen bonds linking SQA and SQB to the bRC protein environment were investigated by 14,15N and 1H pulsed EPR characterizations of the hfi and nqi tensors.26,27,31–33 The

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C hfi tensors were also determined for the carbons of the 2- and 3-methoxy groups in

both bRC SQs.30 Select parameters from these studies are summarized in Table 1 for comparison with the values determined for SQLM in this work. A comparative analysis of the hfi and nqi tensors for the histidine Nδ H-bond donors of SQs in different quinone sites has previously found a remarkably good linear correlation between the isotropic hfi constant a(14N) and asymmetry parameter η of the nqi tensor (Figure 5).26 In contrast to K, a(14N) and η of SQLM are significantly different from the corresponding SQA


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values for the bRC. However, the changes in a(14N) and η still retain the previously established linear correlation. This correlation between a(14N) and η in Figure 5 reflects a variation in the hydrogen bond O···H distance and a conserved binding geometry of the SQs with respect to their histidine Nδ H-bond donors in the quinone sites. Thus, the

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N and 1H

ESEEM data for SQLM taken as a whole indicate that the conformation of the His-M219 Nδ – SQ complex is retained in the LM dimer, but with an increased O···H distance.

Figure 5.

Correlation between η and the isotropic hyperfine constant a(14N) for the histidine Nδ in

semiquinone bound proteins. The agreement of the point for SQLM (cyan) with the observed linear correlation (dotted line) supports the conclusion that the binding conformation of the His-M219 Nδ – SQLM complex is similar to that of SQA.

A second hydrogen bonded proton to SQLM is not resolved in either the 1H HYSCORE or four-pulse ESEEM spectra. This can be interpreted as an absence of the second H-bond, or that it possesses an anisotropic coupling less than 2.9 MHz, which is the limit below which a cross-ridge is no longer resolved off of the anti-diagonal in X-band HYSCORE. On the other hand, the existence of cross-peak 4 in the 14N HYSCORE shows that there is another nitrogen interacting with the semiquinone, possessing a(14N) ~ 0.8-1.7 MHz. Based on our prior knowledge of the bRC, we assign this nitrogen to Np of Ala-M260, where a(14N) was found 11 ACS Paragon Plus Environment


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to be 2.6 MHz for SQA. The breadth of cross-peak 4 suggests a spread in the Ala-M260 NpH···O bond length and/or angle. The second hydrogen bond of SQLM with NP is apparently significantly weakened upon removal of the H subunit, and likely heterogeneous and transient in nature. In addition to the data discussed above we performed similar measurements up to 120 K and did not find significant variations in the spectra and relaxation times of the SQLM, indicating an absence in quinone and protein mobility within the temperature range 90-120 K. Thus, the changes of the spectral lineshape and intensity observed in the ESEEM spectra for the SQLM in the LM dimer in comparison with the SQA in bRC result from static disorder of the quinone molecule as a consequence of the weaker binding interactions between the SQ and protein environment. To provide a more quantitative analysis of the structural changes in the LM dimer, DFT calculations have been performed based on a modified model of the SQA described previously.26 Starting from the optimized SQA structure in the bRC we explored the effect of increasing hydrogen bond lengths on the proton TH values and the 14N isotropic hfi constants for both His-M219 and Ala-M260. For His-M219, an elongation of the H-bond to 1.75 Å is required to replicate the experimental TH value (Figure S9), and 1.95 Å is required to reproduce the experimental

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N hfi coupling (Figure S10). Compared to previous

calculations,26 this suggests the His-M219 Nδ – SQLM bond length has increased by ~0.25 Å upon removal of the H subunit. For Ala-M260, lengthening the hydrogen bond to at least 2.05 Å (Figure S9) is necessary to obtain TH ≤ 2.9 MHz. The experimentally determined 0.8-1.7 MHz range for the Ala-M260 Np isotropic hfi coupling corresponds to a 2.15-1.85 Å hydrogen bond length range (Figure S10). These two types of calculations suggest a consistent ~0.4 Å elongation of the Ala-M260 Np – SQLM hydrogen bond. It should be noted that changes in bond angle can also alter the values of the coupling constants significantly,


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and these are not accounted for here where only changes in the H-bond length are investigated. Pulsed ENDOR spectra for SQLM show a slight decrease of the C5‘-methyl proton isotropic coupling, indicating a further increase of the asymmetry in the spin density distribution in the SQLM compared with SQA. This corresponds to an even greater weakening of the Ala-M260 Np – SQLM hydrogen bond than for the His-M219 Nδ – SQLM hydrogen bond (Figure S11 and discussion in the SI), in agreement with the HYSCORE and DFT analyses described above. In summary, pulsed EPR measurements have revealed the structural features of the primary semiquinone in the LM dimer. The His-M219 Nδ – SQLM hydrogen bond is elongated, but with a preserved binding conformation between the imidazole ring and SQ. The Ala-M260 Np – SQLM hydrogen bond is elongated even further and probably possesses a distribution of bond lengths and bond angles (Np – H – O4). The 13C isotope labelling experiment suggests a greater degree of rotational freedom for the methoxy groups of the SQLM, consistent with the overall weakening of SQ binding in the LM dimer. These results allow us to propose a similar influence of the H subunit removal on the quinone-protein interactions in the QB site. The weaker hydrogen bonding and more disordered orientation of the 2-methoxy group likely alters the quinone redox potential difference required for ET between the QA and QB sites. On the other hand, the failure of ET from SQA to QB in the LM dimer gives an additional motivation to conjugate the LM dimer to an electrode surface, as the electrode does not have to compete with QB for the electron from SQA. The linkage of the LM dimer to an electrode surface can be achieved by strategically placed Cys residues, which allow attachment to gold34 or graphite electrodes.35 In principle, the absence of the H subunit could greatly shorten the electron tunneling distance and lead to enhanced photocurrents.



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ASSOCIATED CONTENT Supporting Information. Experimental section, additional details of the experiments and theoretical calculations. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *

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

*

E-mail: [email protected] (P.J.O.).

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT This work was supported by NSF Grant MCB-0818121 (C.A.W.), DE-FG02-08ER15960 Grant (S.A.D.) from Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Sciences, US Department of Energy, and NCRR/NIH Grants S10-RR15878 and S10-RR025438 for pulsed EPR instrumentation. PJOM acknowledges the use of computer resources granted by the EPSRC UK national service for computational chemistry software (NSCCS). A.T.T. gratefully acknowledges support as a NIH trainee of the Molecular Biophysics Training Program (5T32-GM008276). The authors would like to thank Dr. Neal Woodbury for proofreading the manuscript.


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