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Characterization of the Exchangeable Protons in the Immediate Vicinity of the Semiquinone Radical at the QH Site of the Cytochrome bo3 from Escherichia coli

2006; Elsevier BV; Volume: 281; Issue: 25 Linguagem: Inglês

10.1074/jbc.m602544200

1083-351X

Lai Lai Yap, Rimma I. Samoilova, Robert B. Gennis, Sergei A. Dikanov,

Free Radicals and Antioxidants

The cytochrome bo3 ubiquinol oxidase from Escherichia coli resides in the bacterial cytoplasmic membrane and catalyzes the two-electron oxidation of ubiquinol-8 and four-electron reduction of O2 to water. The one-electron reduced semiquinone forms transiently during the reaction, and the enzyme has been demonstrated to stabilize the semiquinone. Two-dimensional electron spin echo envelope modulation has been applied to explore the exchangeable protons involved in hydrogen bonding to the semiquinone by substitution of 1H2Oby 2H2O. Three exchangeable protons possessing different isotropic and anisotropic hyperfine couplings were identified. The strength of the hyperfine interaction with one proton suggests a significant covalent O–H binding of carbonyl oxygen O1 that is a characteristic of a neutral radical, an assignment that is also supported by the unusually large hyperfine coupling to the methyl protons. The second proton with a large anisotropic coupling also forms a strong hydrogen bond with a carbonyl oxygen. This second hydrogen bond, which has a significant out-of-plane character, is from an NH2 or NH nitrogen, probably from an arginine (Arg-71) known to be in the quinone binding site. Assignment of the third exchangeable proton with smaller anisotropic coupling is more ambiguous, but it is clearly not involved in a direct hydrogen bond with either of the carbonyl oxygens. The results support a model that the semiquinone is bound to the protein in a very asymmetric manner by two strong hydrogen bonds from Asp-75 and Arg-71 to the O1 carbonyl, while the O4 carbonyl is not hydrogen-bonded to the protein. The cytochrome bo3 ubiquinol oxidase from Escherichia coli resides in the bacterial cytoplasmic membrane and catalyzes the two-electron oxidation of ubiquinol-8 and four-electron reduction of O2 to water. The one-electron reduced semiquinone forms transiently during the reaction, and the enzyme has been demonstrated to stabilize the semiquinone. Two-dimensional electron spin echo envelope modulation has been applied to explore the exchangeable protons involved in hydrogen bonding to the semiquinone by substitution of 1H2Oby 2H2O. Three exchangeable protons possessing different isotropic and anisotropic hyperfine couplings were identified. The strength of the hyperfine interaction with one proton suggests a significant covalent O–H binding of carbonyl oxygen O1 that is a characteristic of a neutral radical, an assignment that is also supported by the unusually large hyperfine coupling to the methyl protons. The second proton with a large anisotropic coupling also forms a strong hydrogen bond with a carbonyl oxygen. This second hydrogen bond, which has a significant out-of-plane character, is from an NH2 or NH nitrogen, probably from an arginine (Arg-71) known to be in the quinone binding site. Assignment of the third exchangeable proton with smaller anisotropic coupling is more ambiguous, but it is clearly not involved in a direct hydrogen bond with either of the carbonyl oxygens. The results support a model that the semiquinone is bound to the protein in a very asymmetric manner by two strong hydrogen bonds from Asp-75 and Arg-71 to the O1 carbonyl, while the O4 carbonyl is not hydrogen-bonded to the protein. Cytochrome bo3 (cyt bo3) 3The abbreviations used are: cyt bo3, cytochrome bo3 ubiquinol oxidase from E. coli; SQ, semiquinone; QH, the high affinity quinone-binding site; ESE, electron spin echo; ESEEM, electron spin echo envelope modulation; HYSCORE, hyperfine sublevel correlation; ENDOR, electron-nuclear double resonance; 1D, one-dimensional; 2D, two-dimensional; DFT, density functional theory. 3The abbreviations used are: cyt bo3, cytochrome bo3 ubiquinol oxidase from E. coli; SQ, semiquinone; QH, the high affinity quinone-binding site; ESE, electron spin echo; ESEEM, electron spin echo envelope modulation; HYSCORE, hyperfine sublevel correlation; ENDOR, electron-nuclear double resonance; 1D, one-dimensional; 2D, two-dimensional; DFT, density functional theory. is a terminal oxidase in the aerobic respiratory chain of Escherichia coli. It catalyzes the two-electron oxidation of ubiquinol-8 with a semiquinone (SQ) intermediate in an overall reaction that releases two protons to solution. The available evidence suggests that the cyt bo3 ubiquinol oxidase has two Q binding sites (1Sato-Watanabe M. Mogi T. Ogura T. Kitagawa T. Miyoshi H. Iwamura H. Anraku Y. J. Biol. Chem. 1994; 269: 28908-28912Abstract Full Text PDF PubMed Google Scholar, 2Sato-Watanabe M. Mogi T. Sakamoto K. Miyoshi H. Anraku Y. Biochemistry. 1998; 37: 12744-12752Crossref PubMed Scopus (28) Google Scholar, 3Musser S.M. Stowell M.H.B. Lee H.K. Rumbley J.N. Chan S.I. Biochemistry. 1997; 36: 894-902Crossref PubMed Scopus (44) Google Scholar): a low affinity site (QL) where the substrate quinol is oxidized and the product is released, and a high affinity site where the bound quinone species acts as a conduit for electrons, similar to the role of the QA site in the bacterial reaction center (3Musser S.M. Stowell M.H.B. Lee H.K. Rumbley J.N. Chan S.I. Biochemistry. 1997; 36: 894-902Crossref PubMed Scopus (44) Google Scholar, 4Sato-Watanabe M. Mogi T. Miyoshi H. Anraku Y. Biochemistry. 1998; 37: 5355-5361Google Scholar, 5Osborne J.P. Musser S.M. Schultz B.E. Edmondson D.E. Chan S.I. Gennis R.B. Oxygen Homeostasis and Its Dynamics. 1998; (Springer-Verlag, Tokyo): 33-39Crossref Google Scholar, 6Puustinen A. Verkhovsky M.I. Morgan J.E. Belevich N.P. Wikström M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1545-1548Crossref PubMed Scopus (69) Google Scholar, 7Mogi T. Sato-Watanabe M. Miyoshi H. Orii Y. FEBS Lett. 1999; 457: 223-226Crossref PubMed Scopus (7) Google Scholar, 9Schultz B.E. Edmondson D.E. Chan S.I. Biochemistry. 1998; 37: 4160-4168Crossref PubMed Google Scholar). The substrate (QH2) site, referred to as the low affinity site (QL), is equilibrated with the quinone pool in the membrane. The high affinity quinone-binding site (QH), from which Q is not readily removed, stabilizes the SQ. The quinone bound at the QH site functions as a tightly bound cofactor. Pulse radiolysis studies (10Kobayashi K. Tagawa S. Mogi T. Biochemistry. 2000; 39: 15620-15625Crossref PubMed Scopus (29) Google Scholar) have shown that the tightly bound quinone can be rapidly reduced to the semiquinone species and that the tightly bound quinone is essential for rapid electron transfer to heme b. The first order rate constant for the reduction of heme b is 1.5 × 103 s–1, which is approximately the turnover rate of the enzyme. It is reasonably assumed that there is a rapid (>104 s–1) two-electron reduction of QH by the bound substrate QLH2, followed by two one-electron intramolecular transfers from the QHH2 to heme b. Hence, the suggested electron transfer sequence is as in Reaction 1. QLH2⇆QH⇆heme b⇆heme o3-CuB→O2REACTION 1 The function of the quinone cofactor bound at the QH site as a two-electron/one-electron transformer is supported by the electrochemical studies, which show that the QH site stabilizes the SQ form of the quinone bound at this site. The midpoint potential for the two-electron reduction of QHH2 is ∼+100 mV at pH 7.5, and the pH dependence (up to pH 9) of the midpoint potential shows that two protons are taken up by the protein upon reduction, probably forming the protonated dihydroubiquinol: 2 e–1 + 2H+ + QH → QHH2.The midpoint potential for the one-electron oxidation of QHH2 to the SQ species is ∼–13 mV, and the pH dependence of the oxidation of the SQ to the fully oxidized quinone indicates oxidation to form the SQ is coupled to the loss of a proton from a group with a pKa of ∼7.5. This has been interpreted as the pKa of the SQ, predicting that the SQ is an anion (QH_˙) above pH 7.5 but is a neutral species (QH_˙H) below pH 7.5 (11Ingledew W.J. Ohnishi T. Salerno J.C. Eur. J. Biochem. 1995; 227: 903-908Crossref PubMed Scopus (70) Google Scholar).The x-ray structure of cyt bo3 (12Abramson J. Riistama S. Larsson G. Jasaitis A. Svensson-Ek. M. Laakkonen L. Puustinen A. Iwata S. Wikström M. Nat. Struct. Biol. 2000; 7: 910-917Crossref PubMed Scopus (353) Google Scholar) does not contain any bound quinone, but site-directed mutagenesis studies (12Abramson J. Riistama S. Larsson G. Jasaitis A. Svensson-Ek. M. Laakkonen L. Puustinen A. Iwata S. Wikström M. Nat. Struct. Biol. 2000; 7: 910-917Crossref PubMed Scopus (353) Google Scholar, 13Hellwig P. Yano T. Ohnishi T. Gennis R.B. Biochemistry. 2002; 41: 10675-10679Crossref PubMed Scopus (33) Google Scholar, 14Hellwig P. Barquera B. Gennis R.B. Biochemistry. 2001; 40: 1077-1082Crossref PubMed Scopus (23) Google Scholar, 15Hellwig P. Mogi T. Tomson F.L. Gennis R.B. Iwata J. Miyoshi H. Mantele W. Biochemistry. 1999; 38: 14683-14689Crossref PubMed Scopus (49) Google Scholar) have identified residues that influence the SQ, resulting in a model for the QH binding site (12Abramson J. Riistama S. Larsson G. Jasaitis A. Svensson-Ek. M. Laakkonen L. Puustinen A. Iwata S. Wikström M. Nat. Struct. Biol. 2000; 7: 910-917Crossref PubMed Scopus (353) Google Scholar). This model (12Abramson J. Riistama S. Larsson G. Jasaitis A. Svensson-Ek. M. Laakkonen L. Puustinen A. Iwata S. Wikström M. Nat. Struct. Biol. 2000; 7: 910-917Crossref PubMed Scopus (353) Google Scholar) proposes hydrogen bonds from Asp-75 and Arg-71 to one of the carbonyl oxygens and hydrogen bonds from Gln-101 and His-98 to the second carbonyl oxygen.Information about the electronic structure of the semiquinone radical in cyt bo3 and its interaction with the protein environment is available from high resolution EPR studies. The hydrogen-bonded protons around the SQ in cyt bo3 were previously examined using electron-nuclear double resonance (ENDOR) techniques in conjunction with deuterium (D2O) exchange. The Q-band ENDOR spectrum showed only one splitting from exchangeable deuterium consistent with hydrogen bonding to the quinone oxygen(s) (16Veselov A.V. Osborne J.P. Gennis R.B. Scholes C.P. Biochemistry. 2000; 39: 3169-3175Crossref PubMed Scopus (30) Google Scholar). Two pairs of exchange-sensitive features were observed in the X-band ENDOR spectra of cyt bo3 bound to either native quinone or to exogenously added quinones (17Hastings S.F. Heathcote P. Ingledew W.J. Rigby S.E.J. Eur. J. Biochem. 2000; 267: 5638-5645Crossref PubMed Scopus (19) Google Scholar, 18McMillan F. Grimaldi S. Ostermann T. Weiden N. Mogi T. Ludwig B. Miyoshi H. Michel H. Prisner T.F. 5th Meeting of the European Federation of EPR Groups. 2003; (2003): 7-11Google Scholar). The data were interpreted by different laboratories as indicating either one H-bonded proton (18McMillan F. Grimaldi S. Ostermann T. Weiden N. Mogi T. Ludwig B. Miyoshi H. Michel H. Prisner T.F. 5th Meeting of the European Federation of EPR Groups. 2003; (2003): 7-11Google Scholar) or two equivalent H-bonded protons (17Hastings S.F. Heathcote P. Ingledew W.J. Rigby S.E.J. Eur. J. Biochem. 2000; 267: 5638-5645Crossref PubMed Scopus (19) Google Scholar).A multifrequency (9, 34, and 94 GHz) EPR study was performed with cyt bo3 bound to ubiquinone-2 selectively labeled with 13C at either the 1- or the 4-carbonyl carbons (19Grimaldi S. Ostermann T. Weiden N. Mogi T. Miyoshi H. Ludwig B. Michel H. Prisner T.F. MacMillan F. Biochemistry. 2003; 42: 5632-5639Crossref PubMed Scopus (46) Google Scholar). The EPR spectra revealed significant differences in the hyperfine tensors of these carbons indicating that the radical interacts with the protein in a highly asymmetric manner (19Grimaldi S. Ostermann T. Weiden N. Mogi T. Miyoshi H. Ludwig B. Michel H. Prisner T.F. MacMillan F. Biochemistry. 2003; 42: 5632-5639Crossref PubMed Scopus (46) Google Scholar), consistent with a strong hydrogen bond to the O1 carbonyl oxygen. In addition, one- and two-dimensional electron spin echo envelope modulation (1D and 2D ESEEM) studies have shown that the SQ in cyt bo3 forms an H-bond with a 14N atom from the protein environment (20Grimaldi S. MacMillan F. Ostermann T. Ludwig T. Michel H. Prisner T. Biochemistry. 2001; 40: 1037-1043Crossref PubMed Scopus (36) Google Scholar), tentatively assigned either as coming from the polypeptide backbone (20Grimaldi S. MacMillan F. Ostermann T. Ludwig T. Michel H. Prisner T. Biochemistry. 2001; 40: 1037-1043Crossref PubMed Scopus (36) Google Scholar) or from an arginine side chain (19Grimaldi S. Ostermann T. Weiden N. Mogi T. Miyoshi H. Ludwig B. Michel H. Prisner T.F. MacMillan F. Biochemistry. 2003; 42: 5632-5639Crossref PubMed Scopus (46) Google Scholar).Thus, previous studies have established the presence of at least one H-bond between the SQ in cyt bo3 and a nitrogen that is part of the protein environment. These results do not exclude additional hydrogen bonds between the quinone oxygens and other protein H-bonding partners, including S and O atoms.The current work provides additional information about the exchangeable protons in hydrogen bonds to the SQ species in cyt bo3 by using a combination of 1D and 2D ESEEM, and pulsed ENDOR. The major new observation is that there are at least three exchangeable protons with distinct hyperfine couplings in the immediate environment of the SQ. The data support a model in which the semiquinone is stabilized by strong hydrogen bonds from both Asp-75 and Arg-71 to oxygen O1 and with no hydrogen bonds to carbonyl O4, resulting in a highly asymmetric spin distribution.EXPERIMENTAL PROCEDURESSample Preparation—The pJRHisA plasmid encoding wild-type cyt bo3 was transformed into the C43(DE3) E. coli strain (Avidis, France). Cells were grown in LB medium containing 100 μg/ml ampicillin, 0.3% lactic acid, and 500 μm CuSO4 at 37°C and harvested at the mid-logarithm phase. Harvested cells were resuspended in 50 mm K2HPO4, 5 mm MgSO4, pH 8.3, and broken by passing through a microfluidizer (Microfluidics Corp., Worcester, MA) at 10,000 p.s.i. (three times), followed by centrifugation at 16,000 × g for 30 min to remove cell debris. Membranes were then isolated from the supernatant by centrifugation at 180,000 × g for at least 5 h. The isolated membranes were suspended in 50 mm K2HPO4, pH 8.3, and solubilized with 1% n-dodecyl β-d-maltoside (Anatrace, OH) by stirring at 4°C for 2 h. The unsolubilized material was removed by centrifugation at 15,000 × g for 1 h. The solubilized cyt bo3 was loaded onto a nickel-nitrilotriacetic acid column and purified as described previously (21Rumbley J.N. Furlong Nickels E. Gennis R.B. Biochem. Biophys. Acta. 1997; 1340: 131-142Crossref PubMed Scopus (77) Google Scholar). The purified protein was then dialyzed overnight in 50 mm K2HPO4, 0.1% n-dodecyl β-d-maltoside, pH 8.3, and concentrated to ∼400 μm. For the deuterated sample, the dialyzed protein was concentrated, exchanged with deuterated 50 mm K2HPO4, 0.1% n-dodecyl β-d-maltoside, pD 8.3, and further concentrated to ∼400 μm. The enzyme was anaerobically reduced under an argon atmosphere with 500-times excess sodium ascorbate, and the reduced sample was then transferred to an argon-flushed EPR tube, followed by rapid freezing in liquid nitrogen.EPR Measurements—The continuous wave and pulsed EPR experiments were carried out using X-band Bruker ELEXSYS E580 spectrometers equipped with Oxford CF 935 cryostats. Unless otherwise indicated, all measurements were made at 50 K. Several types of ESE experiments with different pulse sequences were employed with appropriate phase-cycling schemes to eliminate unwanted features from experimental echo envelopes. Among them are two-pulse and four-pulse sequences. In the two-pulse experiment (π/2-τ-π-τ-echo) the intensity of the echo signal is measured as a function of the time interval τ between two microwave pulses with turning angles π/2 and π to generate an echo envelope that maps the time course of relaxation of the spin system (in ESEEM), or as a function of magnetic field at fixed τ (in field-sweep ESE). In the 2D four-pulse ESEEM experiment (π/2-τ-π/2-t1-π-t2-π/2-τ-echo), also called HYSCORE (22Höfer P. Grupp A. Nebenführ H. Mehring M.M. Chem. Phys. Lett. 1986; 132: 279-284Crossref Scopus (503) Google Scholar), the intensity of the stimulated echo after the fourth pulse is measured with t2 and t1 varied, and τ constant. Such a 2D set of echo envelopes gives, after complex Fourier transformation, a 2D spectrum with equal resolution in each direction. Spectral processing of ESEEM patterns was performed using Bruker WIN-EPR software.Pulsed ENDOR spectra of the radical in cyt bo3 were obtained using Davies (π-t-π/2-τ-π-τ) and Mims (π/2-τ-π/2-t-π/2-τ) sequences with different pulse lengths. In addition, radio frequency π pulse is applied during the time interval t in both sequences. The specifics of these experiments are described in detail elsewhere (23Schweiger A. Jeschke G. Principles of Pulse Electron Paramagnetic Resonance. 2001; (Oxford University Press): 359-405Google Scholar).Characteristics of HYSCORE Spectra from I = ½ Nuclei—The most informative experimental data regarding the ligand environment of the semiquinone were obtained from the 2D ESEEM (HYSCORE) experiment (22Höfer P. Grupp A. Nebenführ H. Mehring M.M. Chem. Phys. Lett. 1986; 132: 279-284Crossref Scopus (503) Google Scholar). The basic advantage of the HYSCORE technique is the creation of 2D spectra with off-diagonal cross-peaks (να, νβ) and (νβ, να), whose coordinates are nuclear frequencies from opposite electron spin manifolds. The cross-peaks simplify significantly the analysis of congested spectra by correlating and spreading out the nuclear frequencies. In addition, the HYSCORE experiment separates overlapping peaks along a second dimension and enhances the signal-to-noise ratio through a second Fourier transform. HYSCORE is also valuable for the detection of extended anisotropic peaks of low intensity, which are not seen in 1D ESEEM spectra.Orientationally disordered (i.e. powder) spectra of I = ½ nuclei also reveal, in the form of cross-peak contour projections, the interdependence between να and νβ values in the same orientation. Analysis of the contours allows for direct, simultaneous determination of the nuclear isotropic and anisotropic hyperfine coupling constants (24Dikanov S.A. Bowman M.K. J. Magn. Reson. A. 1995; 116: 125-128Crossref Scopus (109) Google Scholar) (see "Appendix").RESULTSEPR and ESEEM Spectra of the Semiquinone—The field-sweep ESE spectrum of cyt bo3 recorded at 50 K (Fig. 1) shows only one line from the SQ with g ∼2.0047 and the width ∼1.2 millitesla at the half-height. Some measurements were also performed at 90 K, and the results were similar to those obtained at 50 K.The 1D and 2D ESEEM spectra of the SQ at frequencies <10 MHz, appropriate for 14N nuclei, are identical to those reported previously by Grimaldi et al. (20Grimaldi S. MacMillan F. Ostermann T. Ludwig T. Michel H. Prisner T. Biochemistry. 2001; 40: 1037-1043Crossref PubMed Scopus (36) Google Scholar). These spectra were used to determine the quadrupole coupling constant K = e2qQ/4h = 0.93 MHz, and the asymmetry parameter η = 0.51, which are consistent with assigning this to a 14N nitrogen from the NH or NH2 groups forming an H-bond with the SQ (see "Discussion").Proton HYSCORE—Fig. 2 (A–C) shows the proton part of the HYSCORE spectrum (τ = 136 ns) of the SQ radical in the cyt bo3 sample prepared in 1H2O buffer. In addition to a diagonal peak with extended shoulders at the proton Zeeman frequency (νH ∼14.7 MHz), the spectra in Fig. 2 contain up to six pairs of resolved cross-peaks located symmetrically relative to the diagonal. They are designated 1, 2, 2′,3,3′, and 4. The cross-peaks labeled 1 demonstrate the largest hyperfine splitting, of the order ∼10 MHz. The cross-peaks 2 possess the most extended anisotropic contour in the area further up the diagonal. The peaks 2′,3, 3′, and 4 are located in a similar area of the plot close to each other and partially overlap at the low intensity levels. The contours of these peaks could be separated at the higher levels of the intensity, as shown in Fig. 2C, and used for quantitative analysis. The 2, 2′, 3, and 3′ contours deviate from the normal to the diagonal, indicating a significant anisotropic component. In contrast, contours 1 and 4 are approximately normal to the diagonal, suggesting smaller anisotropy. Cross-peaks 2–4 (Fig. 2D) completely disappeared in the HYSCORE spectra obtained under the same conditions using the sample with 2H2O, showing that these are produced by exchangeable protons. However, cross-peaks 1 and the diagonal peak, with its shoulders, still appear in the spectra.FIGURE 2The proton part of the HYSCORE spectra of the SQ at the QH site of cyt bo3. A, C, and D, contour presentations of the spectra used for the quantitative analysis of lineshapes; B, stacked presentation showing more clearly the relative intensities and shapes of the peaks. Spectrum C shows higher levels of intensity than spectrum A. The microwave frequency was 9.68 GHz, the magnetic field was 345.0 milliteslas, and the time τ between first and second microwave pulses was 136 ns. Spectra were obtained after Fourier transformation of 2D time-domain patterns containing 256 × 256 points with a step of 20 ns.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Quantitative analysis of the cross-peak contour lineshapes (see "Appendix") finds that cross-peaks 2 and 2′ are produced by the same proton (H2) and that cross-peaks 3 and 3′ are also from the same proton (H3). Hence, these data derive from four protons coupled to the SQ: H1, H2, H3, and H4. Among them, H2, H3, and H4 are exchangeable protons. The isotropic (a) and anisotropic (T) components of the axial hyperfine tensors for these protons are summarized in Table 1.TABLE 1Hyperfine tensors of the protons H1–H4 derived from HYSCORE spectraProtona and TA⊥ = a - TA∥ = a + 2TMHzH110.0, 1.78.313.4H2±0.7, ∓6.3±7.0∓11.9H3∓1.2, ±4.2∓5.4±7.2H4∓4.6, ±1.7∓6.3∓1.2 Open table in a new tab Pulsed ENDOR—Fig. 3 shows Davies pulsed ENDOR spectra for the radical in QH site of cyt bo prepared in 1H2O and 2H2O. These spectra were obtained with two different lengths tp of the inverting π pulse. The spectrum recorded with tp = 240 ns shows complex overlap of the lines in the middle of the spectrum around the proton Zeeman frequency (νH). It is seen that the shoulders of this complex line decrease after 1H/2H exchange. The intensity from the weakly coupled protons around the proton Zeeman frequency is suppressed in the spectra recorded with shorter duration tp = 64 ns. The spectrum in 1H2O contains two pairs of peaks located symmetrically relative to the νH with splittings of ∼11 MHz and ∼5 MHz. The pair of peaks with the smaller splitting is absent in the spectrum of the sample prepared in 2H2O, thus indicating their assignment to the exchangeable proton(s). In contrast, a new doublet with splitting ∼0.73 MHz, centered at the deuterium Zeeman frequency (νD) and corresponding to the proton splitting ∼5 MHz, appears in these spectra (Fig. 3). One can also note that the shape of the peaks with ∼11 MHz splitting also changes in this spectrum due to overlap with some spectral features from exchangeable protons. The shape of these peaks after deuterium substitution becomes more asymmetric, allowing us to specify parallel and perpendicular canonical frequencies with the splittings |A##| ∼10 MHz and |A∥| ∼13 MHz. These features correspond to the cross-peaks from H1 in the HYSCORE spectra, and they are assigned to the methyl protons with the hyperfine couplings shown in Table 1 (see also "Appendix").FIGURE 3Pulsed ENDOR spectra obtained using Davies ENDOR sequence of the SQ at the QH site of cyt bo3 prepared in 1 H2O(a and c) and 2H2O (b and d). Length of the first inverting microwave π pulse is 240 ns for a and b, and 64 ns for c and d. Inset, Mims ENDOR spectrum of the SQ in 1H2O(f) and 2H2O(g).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The current data can be compared with the results from previous studies. Hastings et al. (17Hastings S.F. Heathcote P. Ingledew W.J. Rigby S.E.J. Eur. J. Biochem. 2000; 267: 5638-5645Crossref PubMed Scopus (19) Google Scholar) reported two splittings |A##| = 10.8 MHz and |A∥| = 14.4 MHz (corresponding to a = 12 MHz and T = 1.2 MHz) that were not sensitive to deuterium exchange in X-band ENDOR spectra of decyl-ubiquinone in the QH site of cyt bo3. These peaks were assigned to the protons of the methyl substituent. MacMillan et al. (18McMillan F. Grimaldi S. Ostermann T. Weiden N. Mogi T. Ludwig B. Miyoshi H. Michel H. Prisner T.F. 5th Meeting of the European Federation of EPR Groups. 2003; (2003): 7-11Google Scholar) found |A##| = 9.3 MHz and |A∥| = 13.0 MHz (a = 10.5 MHz and T = 1.2 MHz) for these protons by pulsed X-band ENDOR of native ubiquinone-8 in cyt bo3. The most significant loss of intensity after 1H/2H exchange was reported by these authors at frequencies corresponding to the couplings |A##| = 5.1 MHz and |A∥| = 11.7 MHz (17Hastings S.F. Heathcote P. Ingledew W.J. Rigby S.E.J. Eur. J. Biochem. 2000; 267: 5638-5645Crossref PubMed Scopus (19) Google Scholar) and |A##| = 4.5 MHz and |A∥| = 9.1 MHz (18McMillan F. Grimaldi S. Ostermann T. Weiden N. Mogi T. Ludwig B. Miyoshi H. Michel H. Prisner T.F. 5th Meeting of the European Federation of EPR Groups. 2003; (2003): 7-11Google Scholar). In both of these studies, the features were assigned to the same proton(s) and used for the calculation of the anisotropic component of the hyperfine tensor and H-bond length.Veselov et al. (16Veselov A.V. Osborne J.P. Gennis R.B. Scholes C.P. Biochemistry. 2000; 39: 3169-3175Crossref PubMed Scopus (30) Google Scholar) performed orientation-selected Q-band ENDOR experiments with the native SQ in the QH site of cyt bo3. They reported the isotropic coupling of the methyl protons to be ∼11 MHz and a loss of intensity in the 4- to 5-MHz region around the proton Zeeman frequency after 1H/2H exchange. Accordingly, the deuterium ENDOR spectrum recorded at gy exhibits only one resolved splitting ∼0.8 MHz, corresponding to a proton coupling of 5.2 MHz.In summary, the current work, as well as the previous experiments, have all found a remarkably large anisotropic coupling constant ∼10–11 MHz from the methyl protons. In addition, these studies also show similar exchangeable proton and deuterium ENDOR features. Proton ENDOR spectra show a loss of intensity after 1H/2H exchange, which is most significant at frequencies corresponding to the couplings |A##| = 4.5–5.1 MHz and |A∥| = 9.1–11.7 MHz.The powder ENDOR spectrum simulated with the contribution of all three exchangeable protons H2–H4 using the hyperfine tensors given in Table 1 reproduces intense features with a splitting of ∼5 MHz resulting from the overlap of lines from all three protons and |A∥| peaks with the splitting ∼12 MHz from H2 (Fig. 4, "Appendix"). The shape of these features depends on the linewidth of individual ENDOR transitions. Additional factors that may influence the width of these lines in the spectra are the non-axiality of the hyperfine tensors and "strain" in the values of the hyperfine couplings. Thus, the 2D ESEEM data for protons H2, H3, and H4 can explain the exchangeable features in the ENDOR spectra described above, and those data previously assigned by other authors (17Hastings S.F. Heathcote P. Ingledew W.J. Rigby S.E.J. Eur. J. Biochem. 2000; 267: 5638-5645Crossref PubMed Scopus (19) Google Scholar, 18McMillan F. Grimaldi S. Ostermann T. Weiden N. Mogi T. Ludwig B. Miyoshi H. Michel H. Prisner T.F. 5th Meeting of the European Federation of EPR Groups. 2003; (2003): 7-11Google Scholar) to one or two equivalent H-bonded protons (see "Discussion").FIGURE 4The calculated ENDOR spectra for the protons H2 (a = ±0. 7 MHz, T = ∓6.3 MHz), H3 (a =∓1.2 MHz, T =±4.2 MHz), H4 (a =∓4.6 MHz, T =±1.7 MHz), and sum spectrum. The individual width of the ENDOR transitions was 0.5 MHz.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONA Neutral versus an Anionic Semiquinone—The hyperfine couplings for both the non-exchangeable methyl protons as well for the exchangeable protons obtained from HYSCORE and ENDOR experiments provide several important clues about the nature of the SQ species in cyt bo3. Previous studies (16Veselov A.V. Osborne J.P. Gennis R.B. Scholes C.P. Biochemistry. 2000; 39: 3169-3175Crossref PubMed Scopus (30) Google Scholar, 17Hastings S.F. Heathcote P. Ingledew W.J. Rigby S.E.J. Eur. J. Biochem. 2000; 267: 5638-5645Crossref PubMed Scopus (19) Google Scholar, 18McMillan F. Grimaldi S. Ostermann T. Weiden N. Mogi T. Ludwig B. Miyoshi H. Michel H. Prisner T.F. 5th Meeting of the European Federation of EPR Groups. 2003; (2003): 7-11Google Scholar), along with the current work, have found a = 10–11 MHz for the methyl protons. This is the largest isotropic constant reported for these protons for ubiquinones bound to proteins or in solution. Hyperfine coupling of 5.5–6.5 MHz has been reported for ubiquinone anion-radicals in different solvents (25MacMillan F. Lendzian F. Lubitz W. Magn. Reson. Chem. 1995; 33: 581-593Crossref Scopus (61) Google Scholar, 26Joela H. Kasa S. Lehtovuori P. Bech M. Acta Chem. Scand. 1997; 51: 233-241Crossref PubMed Scopus (21) Google Scholar, 27Samoilova R.I. van Liemt W. Steggerda W.F. Lugtenburg J. Ho