Colin A. Wraight

University of Illinois, Urbana-Champaign, Urbana, Illinois, United States

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Publications (123)505.04 Total impact

  • [Show abstract] [Hide abstract]
    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.
    Journal of Physical Chemistry Letters 10/2015; 6(22). DOI:10.1021/acs.jpclett.5b01851 · 7.46 Impact Factor
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    ABSTRACT: By utilizing a combined pulsed EPR and DFT approach, the high-resolution structure of the secondary quinone acceptor (SQB) was determined for purple bacterial reaction centres. The development of such a technique is crucial towards an understanding of protein-bound semiquinones on the structural level, as (i) membrane protein crystallography typically results in low resolution structures, and (ii) obtaining protein crystals in the semiquinone form is rarely feasible. The SQB hydrogen bond network was investigated with Q- (~34 GHz) and X-band (~9.7 GHz) pulsed EPR spectroscopy on fully deuterated reactions centers from Rhodobacter sphaeroides. Simulations in the SQB g-tensor reference frame provided the principal values and directions of the H-bond hyperfine tensors. Three protons were detected, one with anisotropic tensor component, T = 4.6 MHz, assigned to the histidine NδH of His-L190, and two others with similar anisotropic constants T = 3.2 and 3.0 MHz assigned to the peptide NpH of Gly-L225 and Ile-L224, respectively. Despite the strong similarity in the peptide couplings, all hyperfine tensors were resolved in the Q-band ENDOR spectra. The Euler angles describing the series of rotations that bring the hyperfine tensors into the SQB g-tensor reference frame were obtained by least-squares fitting of the spectral simulations to the ENDOR data. These Euler angles show the locations of the hydrogen bonded protons with respect to the semiquinone. Our geometry optimized model of SQB used in previous DFT work is in excellent agreement with the angular constraints from the spectral simulations, providing the foundation for future joint pulsed EPR and DFT semiquinone structural determinations in other proteins.
    The Journal of Physical Chemistry B 04/2015; 119(18). DOI:10.1021/acs.jpcb.5b03434 · 3.30 Impact Factor
  • Ágnes Maróti · Colin A Wraight · Péter Maróti ·
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    ABSTRACT: The 2nd electron transfer from the primary ubiquinone QA to the secondary ubiquinone QB in the reaction center (RC) from Rhodobacter sphaeroides involves protonated QB- intermediate state whose low pKa makes the direct observation impossible. Here, we replaced the native ubiquinone by low potential rhodoquinone at the QB binding site of the M265IT mutant RC. Because the in situ midpoint redox potential of QA of this mutant was lowered about the same extent (≈ 100 mV) as that of QB upon exchange of ubiquinone by low potential rhodoquinone, the interquinone (QA→QB) electron transfer became energetically favorable. After subsequent saturating flash excitations, a period of two damped oscillation of the protonated rhodosemiquinone was observed. The QBH• was identified by 1) the characteristic band at 420 nm of the absorption spectrum after the 2nd flash and 2) smaller damping of the oscillation at 420 nm (due to the neutral form) than at 460 nm (attributed to the anionic form). The appearance of the neutral semiquinone was restricted to the acidic pH range indicating a functional pKa of less than 5.5, slightly higher than that of the native ubisemiquinone (pKa < 4.5) at pH 7. The analysis of the pH- and temperature dependences of the rates of the 2nd electron transfer supports the concept of pH-dependent pKa of the semiquinone at the QB binding site. The local electrostatic potential is severely modified by the strongly interacting neighboring acidic cluster and the pKa of the semiquinone is in the middle of the pH range of the complex titration. The kinetic and thermodynamic data are discussed according to the proton-activated electron transfer mechanism combined with pH-dependent functional pKa of the semiquinone at the QB site of the RC.
    Biochemistry 03/2015; 54(12). DOI:10.1021/bi501553t · 3.02 Impact Factor
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    ABSTRACT: Ubiquinone forms an integral part of the electron transport chain in cellular respiration and photosynthesis across a vast number of organisms. Prior experimental results have shown that the photosynthetic reaction center (RC) from Rhodobacter sphaeroides is only fully functional with a limited set of methoxy-bearing quinones, suggesting that specific interactions with this substituent are required to drive electron transport and the formation of quinol. The nature of these interactions has yet to be determined. Through parameterization of a CHARMM-compatible quinone force eld and subsequent molecular dynamics simulations of the quinone-bound RC, we have investigated and characterized the protein interactions with the quinones in the QA and QB sites using both equilibrium simulation and thermodynamic integration. In particular, we identify a specic interaction between the 2-methoxy group of ubiquinone in the QB site and the amide nitrogen of GlyL225 that we implicate in locking the orientation of the 2-methoxy group, thereby tuning the redox potential dierence between the quinones occupying the QA and QB sites. Disruption of this interaction leads to weaker binding in a ubiquinone analog that lacks a 2-methoxy group, a finding supported by reverse electron transfer EPR experiments of the QA(-)QB(-) biradical and competitive binding assays.
    Biochemistry 03/2015; 54(12). DOI:10.1021/acs.biochem.5b00033 · 3.02 Impact Factor
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    ABSTRACT: The electrostatic potential in the secondary quinone (QB) binding site of the reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides determines the rate and free energy change (driving force) of electron transfer to QB. It is controlled by the ionization states of residues in a strongly interacting cluster around the QB site. Reduction of the QB induces change of the ionization states of residues and binding of protons from the bulk. Stigmatellin, an inhibitor of the mitochondrial and photosynthetic respiratory chain, has been proven to be a unique voltage probe of the QB binding pocket. It binds to the QB site with high affinity, and the pK value of its phenolic group monitors the local electrostatic potential with high sensitivity. Investigations with different types of detergent as a model system of isolated RC revealed that the pK of stigmatellin was controlled overwhelmingly by electrostatic and slightly by hydrophobic interactions. Measurements showed a high pK value (>11) of stigmatellin in the QB pocket of the dark-state wild-type RC, indicating substantial negative potential. When the local electrostatics of the QB site was modulated by a single mutation, L213Asp→Ala, or double mutations, L213Asp-L212Glu→Ala-Ala (AA), the pK of stigmatellin dropped to 7.5 and 7.4, respectively, which corresponds to a >210 mV increase in the electrostatic potential relative to the wild-type RC. This significant pK drop (ΔpK > 3.5) decreased dramatically to (ΔpK > 0.75) in the RC of the compensatory mutant (AA+M44Asn→AA+M44Asp). Our results indicate that the L213Asp is the most important actor in the control of the electrostatic potential in the QB site of the dark-state wild-type RC, in good accordance with conclusions of former studies using theoretical calculations or light-induced charge recombination assay. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.
    Biophysical Journal 01/2015; 108(2):379-94. DOI:10.1016/j.bpj.2014.11.3463 · 3.97 Impact Factor
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    ABSTRACT: Photosynthetic reaction centers (RCs) from Rb. sphaeroides with a genetically engineered 7-his-tag at the C-terminus of the M-subunit are bound to a Ni-NTA-modified gold surface. Subsequently, the bound RCs are subjected to in situ dialysis in the presence of lipid micelles to form a protein-tethered lipid bilayer membrane (ptBLM). Redox properties of the RC thus immobilized are investigated by cyclic voltammetry. Photocurrrents are generated in the range of 10 μA cm–2, however, different from previous studies at potentials of −200 and −300 mV, and without cytochrome c as a mediator. The unexpected behavior is explained in terms of an interprotein reaction between RC molecules promoted by the lipid bilayer, which we had previously detected by surface-enhanced infrared absorption spectroscopy.
    The Journal of Physical Chemistry C 01/2015; 119(2):150106130737006. DOI:10.1021/jp510006n · 4.77 Impact Factor
  • Ágnes Maróti · Colin A. Wraight · Péter Maróti ·
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    ABSTRACT: The 2nd electron transfer in reaction center of photosynthetic bacterium Rhodobacter sphaeroides is a two step process in which protonation of QB− precedes interquinone electron transfer. The thermal activation and pH dependence of the overall rate constants of different RC variants were measured and compared in solvents of water (H2O) and heavy water (D2O). The electron transfer variants where the electron transfer is rate limiting (wild type and M17DN, L210DN and H173EQ mutants) do not show solvent isotope effect and the significant decrease of the rate constant of the second electron transfer in these mutants is due to lowering the operational pKa of QB−/QBH: 4.5 (native), 3.9 (L210DN), 3.7 (M17DN) and 3.1 (H173EQ) at pH 7. On the other hand, the proton transfer variants where the proton transfer is rate limiting demonstrate solvent isotope effect of pH-independent moderate magnitude (2.11 ± 0.26 (WT + Ni2 +), 2.16 ± 0.35 (WT + Cd2 +) and 2.34 ± 0.44 (L210DN/M17DN)) or pH-dependent large magnitude (5.7 at pH 4 (L213DN)). Upon deuteration, the free energy and the enthalpy of activation increase in all proton transfer variants by about 1 kcal/mol and the entropy of activation becomes negligible in L210DN/M17DN mutant. The results are interpreted as manifestation of equilibrium and kinetic solvent isotope effects and the structural, energetic and kinetic possibility of alternate proton delivery pathways are discussed.
    Biochimica et Biophysica Acta (BBA) - Bioenergetics 11/2014; 1847(2). DOI:10.1016/j.bbabio.2014.11.002 · 5.35 Impact Factor
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    ABSTRACT: Recent studies have shown that only quinones with a 2-methoxy group can act simultaneously as the primary (QA) and secondary (QB) electron acceptors in photosynthetic reaction centers from purple bacteria such as Rb. sphaeroides. (13)C HYSCORE measurements of the 2-methoxy group in the semiquinone states, SQA and SQB, were compared with DFT calculations of the (13)C hyperfine couplings as a function of the 2-methoxy dihedral angle. X-ray structure comparisons support 2-methoxy dihedral angle assignments corresponding to a redox potential gap (ΔE m) between QA and QB of 175-193 mV. A model having a methyl group substituted for the 2-methoxy group exhibits no electron affinity difference. This is consistent with the failure of a 2-methyl ubiquinone analogue to function as QB in mutant reaction centers with a ΔE m of ∼160-195 mV. The conclusion reached is that the 2-methoxy group is the principal determinant of electron transfer from QA to QB in type II photosynthetic reaction centers with ubiquinone serving as both acceptor quinones.
    Journal of Physical Chemistry Letters 08/2014; 5(15):2506-2509. DOI:10.1021/jz500967d · 7.46 Impact Factor
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    ABSTRACT: X- and Q-band pulsed EPR spectroscopy was applied to study the interaction of the Q(A) site semiquinone (SQ(A)) with nitrogens from the local protein environment in natural abundance N-14 and in N-15 uniformly labeled photosynthetic reaction centers of Rhodobacter sphaeroides. The hyperfine and nuclear quadrupole tensors for His-M219 N-delta and Ala-M260 peptide nitrogen (N-p) were estimated through simultaneous simulation of the Q:band N-15 Davies ENDOR, X- and Q-band N-14,N-15 HYSCORE, and X-band N-14 three-pulse ESEEM spectra, with support from DFT calculations. The hyperfine coupling constants were found to be a(N-14) = 2.3 MHz, T = 0.3 MHz for His-M219 N-delta and a(N-14) = 2.6 MHz, T = 0.3 MHz for Ala-M260 N-p. Despite that His-M219 N-delta is established as the stronger of the two H-bond donors, Ala-M260 N-p is found to have the larger value of a(14N). The nuclear quadrupole coupling constants were estimated as e(2)Qq/4h = 0.38 MHz, eta = 0.97 and e(2)Qq/4h = 0.74 MHz, eta = 0.59 for His-M219 N-delta and Ala-M260 N-p, respectively. An analysis of the available data on nuclear quadrupole tensors for imidazole nitrogens found in semiquinone-binding proteins and copper complexes reveals these systems share similar electron occupancies of the protonated nitrogen orbitals. By applying the Townes-Dailey model, developed previously for copper complexes, to the semiquinones, we find the asymmetry parameter eta to be a sensitive probe of the histidine N-delta-semiquinone hydrogen bond strength. This is supported by a strong correlation observed between eta and the isotropic coupling constant a(N-14) and is consistent with previous computational works and our own semiquinone-histidine model calculations. The empirical relationship presented here for a(N-14) and eta will provide an important structural characterization tool in future studies of semiquinone-binding proteins.
    The Journal of Physical Chemistry B 07/2014; 118(31). DOI:10.1021/jp5051029 · 3.30 Impact Factor
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    Melvin Okamura · Colin A. Wraight · Rienk van Grondelle ·
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    ABSTRACT: This Special Issue of Photosynthesis Research honors Louis M. N. Duysens, Roderick K. Clayton, and George Feher, three pioneering researchers whose work on bacterial photosynthesis laid much of the groundwork for our understanding of the role of the reaction center in photosynthetic light energy conversion. Their key discoveries are briefly summarized and an overview of the special issue is presented.
    Photosynthesis Research 05/2014; 120(1-2). DOI:10.1007/s11120-013-9952-9 · 3.50 Impact Factor
  • Chang Sun · Colin A. Wraight ·

    Biophysical Journal 01/2014; 106(2):588a. DOI:10.1016/j.bpj.2013.11.3256 · 3.97 Impact Factor
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    Biophysical Journal 01/2014; 106(2):370a. DOI:10.1016/j.bpj.2013.11.2095 · 3.97 Impact Factor
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    Biophysical Journal 01/2014; 106(2):370a. DOI:10.1016/j.bpj.2013.11.2096 · 3.97 Impact Factor
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    ABSTRACT: The secondary quinone anion radical QB- (SQB) in reaction centers of Rhodobacter sphaeroides interacts with Nδ of His-L190 and Np (peptide nitrogen) of Gly-L225 involved in hydrogen bonds to the QB carbonyls. In this work, S-band (~3.6 GHz) ESEEM was used with the aim of obtaining a complete characterization of the nuclear quadrupole interaction (nqi) tensors for both nitrogens by approaching the cancellation condition between the isotropic hyperfine coupling and 14N Zeeman frequency at lower microwave frequencies. By performing measurements at S-band we found a dominating contribution of Nδ in the form of a zero-field nqi triplet at 0.55 MHz, 0.92 MHz, and 1.47 MHz, defining the quadrupole coupling constant K = e2qQ/4h = 0.4 MHz and associated asymmetry parameter η = 0.69. Estimates of the hyperfine interaction (hfi) tensors for Nδ and Np were obtained from simulations of 1D and 2D 14,15N X-band and three-pulse 14N S-band spectra with all nuclear tensors defined in the SQB g-tensor coordinate system. From simulations we conclude that the contribution of Np to the S-band spectrum is suppressed by its strong nqi and weak isotropic hfi comparable to the level of hyperfine anisotropy, despite the near-cancellation condition for Np at S-band. The excellent agreement between our EPR simulations and DFT calculations of the nitrogen hfi and nqi tensors to SQB is promising for the future application of powder ESEEM to full tensor characterizations.
    The Journal of Physical Chemistry B 01/2014; 118(6). DOI:10.1021/jp411023k · 3.30 Impact Factor
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    Colin A Wraight ·
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    ABSTRACT: Roderick K. Clayton passed away on October 23, 2011, at the age of 89, shortly after the plan for this dedicatory issue of Photosynthesis Research had been hatched. I had just written a lengthy letter to him to re-establish contact after a hiatus of 2 or 3 years, and to suggest that I visit him to talk about his life. It isn't clear whether he saw the letter or not, but it was found at his home in Santa Rosa, California. Fortunately, Rod has written two memoirs for Photosynthesis Research that not only cover much of his research on reaction centers (Photosynth Res 73:63-71, 2002) but also provide a humorous and honest look at his personal life (Photosynth Res 19:207-224, 1988). I cannot hope to improve on these and will try, instead, to fill in some of the gaps that Rod's own writing has left, and offer some of my own personal recollections over the more recent years.
    Photosynthesis Research 11/2013; 120(1-2). DOI:10.1007/s11120-013-9948-5 · 3.50 Impact Factor
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    ABSTRACT: Only quinones with a 2-methoxy group can act simultaneously as the primary (QA) and secondary (QB) electron acceptors in photosynthetic reaction centers from Rb. sphaeroides. 13C HYSCORE measurements of the 2-methoxy in the semiquinone states, SQA and SQB, were compared with QM calculations of the 13C couplings as a function of dihedral angle. X-ray structures support dihedral angle assignments corre-sponding to a redox potential gap (∆Em) between QA and QB of ~180 mV. This is consistent with the failure of a ubiquinone analog lacking the 2-methoxy to func-tion as QB in mutant reaction centers with a ∆Em ≈ 160-195 mV.
    Biochemistry 09/2013; 52(41). DOI:10.1021/bi4011896 · 3.02 Impact Factor
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    ABSTRACT: Surface-enhanced IR absorption spectroscopy (SEIRAS) in the ATR configuration has been performed on reaction centers (RCs) from R. sphaeroides. Surface-enhancement is achieved by a thin, structured gold film present on the surface of an ATR crystal. Purified RCs are immobilized as a monolayer on top of the gold film via a poly his-tag engineered to the C-terminal end of the M subunit. Subsequently, the RCs are reconstituted into a lipid bilayer by in situ dialysis. Light-minus-dark absorbance spectra were recorded under continuous illumination using the spectrum in the dark as the reference. A number of strong bands have been observed indicating the excitation of the special pair as well as alterations of quinone/quinol species. Spectra were recorded at different time intervals with and without liposoluble Q10 coreconstituted into the lipid phase. A steady (photostationary) state was approached slowly and bands were found to increase or decrease reversibly on illumination and relaxation. Tentative assignments were made for some bands, based on previous FTIR measurements. The long time scale of these processes was tentatively explained in terms of interprotein reactions of RC molecules.
    The Journal of Physical Chemistry C 07/2013; 117(32):16357. DOI:10.1021/jp4056347 · 4.77 Impact Factor
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    ABSTRACT: Ubiquinone is an almost universal, membrane-associated redox mediator. Its ability to accept either one or two electrons allows it to function in critical roles in biological electron transport. The redox properties of ubiquinone in vivo are determined by its environment in the binding sites of proteins and by the dihedral angle of each methoxy group relative to the ring plane. This is an attribute unique to ubiquinone among natural quinones and could account for its widespread function with many different redox complexes. In this work, we use the photosynthetic reaction center as a model system for understanding the role of methoxy conformations in determining the redox potential of the ubiquinone/semiquinone couple. Despite the abundance of X-ray crystal structures for the reaction center, quinone site resolution has thus far been too low to provide a reliable measure of the methoxy dihedral angles of the primary and secondary quinones, QA and QB. We performed 2D ESEEM (HYSCORE) on isolated reaction centers with ubiquinones (13)C-labeled at the headgroup methyl and methoxy substituents, and have measured the (13)C isotropic and anisotropic components of the hyperfine tensors. Hyperfine couplings were compared to those derived by DFT calculations as a function of methoxy torsional angle allowing estimation of the methoxy dihedral angles for the semiquinones in the QA and QB sites. Based on this analysis, the orientation of the 2-methoxy groups are distinct in the two sites, with QB more out of plane by 20-30°. This corresponds to an ≈50 meV larger electron affinity for the QB quinone, indicating a substantial contribution to the experimental difference in redox potentials (60-75 mV) of the two quinones. The methods developed here can be readily extended to ubiquinone-binding sites in other protein complexes.
    Biochemistry 06/2013; 52(27). DOI:10.1021/bi400489b · 3.02 Impact Factor
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    Biophysical Journal; 01/2013
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    Biochimica et Biophysica Acta (BBA) - Bioenergetics 10/2012; 1817:S30. DOI:10.1016/j.bbabio.2012.06.091 · 5.35 Impact Factor

Publication Stats

4k Citations
505.04 Total Impact Points


  • 1977-2015
    • University of Illinois, Urbana-Champaign
      • • Center for Biophysics and Computational Biology
      • • Department of Biochemistry
      • • Department of Plant Biology
      • • Department of Microbiology
      Urbana, Illinois, United States
  • 2011-2014
    • The University of Manchester
      • School of Chemistry
      Manchester, England, United Kingdom
  • 2013
    • Boston University
      Boston, Massachusetts, United States
  • 1978-1991
    • Urbana University
      Florida, United States
  • 1987
    • Stanford University
      Palo Alto, California, United States
  • 1974-1975
    • Cornell University
      Ithaca, New York, United States
  • 1971
    • University of Bristol
      • Medical School
      Bristol, England, United Kingdom