FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2036681907> ?p ?o ?g. }

• W2036681907 endingPage "18251" @default.
• W2036681907 startingPage "18241" @default.
• W2036681907 abstract "Cytochrome aa3-600 is one of the principle respiratory oxidases from Bacillus subtilis and is a member of the heme-copper superfamily of oxygen reductases. This enzyme catalyzes the two-electron oxidation of menaquinol and the four-electron reduction of O2 to 2H2O. Cytochrome aa3-600 is of interest because it is a very close homologue of the cytochrome bo3 ubiquinol oxidase from Escherichia coli, except that it uses menaquinol instead of ubiquinol as a substrate. One question of interest is how the proteins differ in response to the differences in structure and electrochemical properties between ubiquinol and menaquinol. Cytochrome bo3 has a high affinity binding site for ubiquinol that stabilizes a ubi-semiquinone. This has permitted the use of pulsed EPR techniques to investigate the protein interaction with the ubiquinone. The current work initiates studies to characterize the equivalent site in cytochrome aa3-600. Cytochrome aa3-600 has been cloned and expressed in a His-tagged form in B. subtilis. After isolation of the enzyme in dodecylmaltoside, it is shown that the pure enzyme contains 1 eq of menaquinone-7 and that the enzyme stabilizes a mena-semiquinone. Pulsed EPR studies have shown that there are both similarities as well as significant differences in the interactions of the mena-semiquinone with cytochrome aa3-600 in comparison with the ubi-semiquinone in cytochrome bo3. Our data indicate weaker hydrogen bonds of the menaquinone in cytochrome aa3-600 in comparison with ubiquinone in cytochrome bo3. In addition, the electronic structure of the semiquinone cyt aa3-600 is more shifted toward the anionic form from the neutral state in cyt bo3. Cytochrome aa3-600 is one of the principle respiratory oxidases from Bacillus subtilis and is a member of the heme-copper superfamily of oxygen reductases. This enzyme catalyzes the two-electron oxidation of menaquinol and the four-electron reduction of O2 to 2H2O. Cytochrome aa3-600 is of interest because it is a very close homologue of the cytochrome bo3 ubiquinol oxidase from Escherichia coli, except that it uses menaquinol instead of ubiquinol as a substrate. One question of interest is how the proteins differ in response to the differences in structure and electrochemical properties between ubiquinol and menaquinol. Cytochrome bo3 has a high affinity binding site for ubiquinol that stabilizes a ubi-semiquinone. This has permitted the use of pulsed EPR techniques to investigate the protein interaction with the ubiquinone. The current work initiates studies to characterize the equivalent site in cytochrome aa3-600. Cytochrome aa3-600 has been cloned and expressed in a His-tagged form in B. subtilis. After isolation of the enzyme in dodecylmaltoside, it is shown that the pure enzyme contains 1 eq of menaquinone-7 and that the enzyme stabilizes a mena-semiquinone. Pulsed EPR studies have shown that there are both similarities as well as significant differences in the interactions of the mena-semiquinone with cytochrome aa3-600 in comparison with the ubi-semiquinone in cytochrome bo3. Our data indicate weaker hydrogen bonds of the menaquinone in cytochrome aa3-600 in comparison with ubiquinone in cytochrome bo3. In addition, the electronic structure of the semiquinone cyt aa3-600 is more shifted toward the anionic form from the neutral state in cyt bo3. A number of prokaryotes contain heme-copper respiratory oxygen reductases, which utilize a membrane-bound quinol as the substrate (electron donor) (1Pereira M.M. Santana M. Teixeira M. Biochim. Biophys. Acta. 2001; 1505: 185-208Crossref PubMed Scopus (387) Google Scholar, 2García-Horsman J.A. Barquera B. Rumbley J. Ma J. Gennis R.B. J. Bacteriol. 1994; 176: 5587-5600Crossref PubMed Scopus (395) Google Scholar). These enzymes (quinol oxidases) are closely related to the cytochrome c oxidases, reduce O2 to water, and also pump protons across the membrane bilayer, generating a proton motive force. The quinol oxidases lack CuA, which is present in the cytochrome c oxidases, and the amino acid sequences of the quinol oxidases can be distinguished from those of the cytochrome c oxidases by the lack of the CuA binding motif. The most intensively studied heme-copper quinol oxidase is the cytochrome bo3 ubiquinol oxidase from Escherchia coli (cyt bo3) 3The abbreviations used are: cyt aa3-600cytochrome aa3-600 menaquinol oxidasecyt bo3cytochrome bo3 ubiquinol oxidase from E. coliSQsemiquinoneQHthe high affinity quinone-binding siteESEEMelectron spin echo envelope modulationHYSCOREhyperfine sublevel correlationENDORelectron-nuclear double resonancehfihyperfine interactionnqinuclear quadrupole interactionDMN2,3-dimethyl-1,4-naphthoquinoneHPCLhigh performance liquid chromatographymTmilliteslaMQmenaquinoneMWmicrowave. (3Lin M.T. Samoilova R.I. Gennis R.B. Dikanov S.A. J. Am. Chem. Soc. 2008; 130: 15768-15769Crossref PubMed Scopus (24) Google Scholar, 4Yap L.L. Samoilova R.I. Gennis R.B. Dikanov S.A. J. Biol. Chem. 2007; 282: 8777-8785Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 5White G.F. Field S. Marritt S. Oganesyan V.S. Gennis R.B. Yap L.L. Katsonouri A. Thomson A.J. Biochemistry. 2007; 46: 2355-2363Crossref PubMed Scopus (10) Google Scholar, 6Yap L.L. Samoilova R.I. Gennis R.B. Dikanov S.A. J. Biol. Chem. 2006; 281: 16879-16887Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 7Kobayashi K. Tagawa S. Mogi T. Biosci. Biotechnol. Biochem. 2009; 73: 1599-1603Crossref PubMed Scopus (3) Google Scholar, 8Matsumoto Y. Murai M. Fujita D. Sakamoto K. Miyoshi H. Yoshida M. Mogi T. J. Biol. Chem. 2006; 281: 1905-1912Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). There are currently over 400 sequences of quinol oxidases that are homologues of cyt bo3. The vast majority of these sequences are from proteobacteria (330 sequences) or the firmicutes (80 sequences). Bacillus subtilis, a firmicute, does not contain ubiquinone but relies on menaquinone (see Fig. 1) as a redox component in its aerobic respiratory chain (9Downey R.J. J. Bacteriol. 1964; 88: 904-911Crossref PubMed Google Scholar). There is a homologue of cyt bo3 in B. subtilis called cytochrome aa3-600, and as expected, this enzyme is a menaquinol oxidase (10Mattatall N.R. Cameron L.M. Hill B.C. Biochemistry. 2001; 40: 13331-13341Crossref PubMed Scopus (8) Google Scholar, 11Lemma E. Schägger H. Kröger A. Arch. Microbiol. 1993; 159: 574-578Crossref PubMed Scopus (22) Google Scholar, 12Fann Y.C. Ahmed I. Blackburn N.J. Boswell J.S. Verkhovskaya M.L. Hoffman B.M. Wikström M. Biochemistry. 1995; 34: 10245-10255Crossref PubMed Scopus (94) Google Scholar, 13Lauraeus M. Wikström M. J. Biol. Chem. 1993; 268: 11470-11473Abstract Full Text PDF PubMed Google Scholar, 14Lauraeus M. Morgan J.E. Wikström M. Biochemistry. 1993; 32: 2664-2670Crossref PubMed Scopus (26) Google Scholar, 15Santana M. Kunst F. Hullo M.F. Rapoport G. Danchin A. Glaser P. J. Biol. Chem. 1992; 267: 10225-10231Abstract Full Text PDF PubMed Google Scholar, 16Villani G. Capitanio N. Bizzoca A. Palese L.L. Carlino V. Tattoli M. Glaser P. Danchin A. Papa S. Biochemistry. 1999; 38: 2287-2294Crossref PubMed Scopus (5) Google Scholar). Whereas E. coli cyt bo3 uses only ubiquinol as a substrate, the B. subtilis cyt aa3-600 is strictly a menaquinol oxidase. The motivation of the current work is to decipher the differences between the protein-quinol interactions of the bo3-type ubiquinol oxidase and the aa3-600 menaquinol oxidase. cytochrome aa3-600 menaquinol oxidase cytochrome bo3 ubiquinol oxidase from E. coli semiquinone the high affinity quinone-binding site electron spin echo envelope modulation hyperfine sublevel correlation electron-nuclear double resonance hyperfine interaction nuclear quadrupole interaction 2,3-dimethyl-1,4-naphthoquinone high performance liquid chromatography millitesla menaquinone microwave. Cyt bo3 has two ubiquinone binding sites, one site with high affinity (QH) and one with low affinity (QL). The QL-site is the substrate binding site, and the quinone at this site exchanges readily with the quinone pool in the membrane (17Welter R. Gu L.Q. Yu L. Yu C.A. Rumbley J. Gennis R.B. J. Biol. Chem. 1994; 269: 28834-28838Abstract Full Text PDF PubMed Google Scholar, 18Sato-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). Despite significant effort (17Welter R. Gu L.Q. Yu L. Yu C.A. Rumbley J. Gennis R.B. J. Biol. Chem. 1994; 269: 28834-28838Abstract Full Text PDF PubMed Google Scholar, 19Sato-Watanabe M. Mogi T. Sakamoto K. Miyoshi H. Anraku Y. Biochemistry. 1998; 37: 12744-12752Crossref PubMed Scopus (28) Google Scholar, 20Ma J. Puustinen A. Wikström M. Gennis R.B. Biochemistry. 1998; 37: 11806-11811Crossref PubMed Scopus (28) Google Scholar), little is known about the location of this site within the protein (21Abramson 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 (358) Google Scholar). Cyt bo3, when purified using the detergent dodecylmaltoside, has 1 eq of ubiquinol-8 bound at the QH-site, and this bound quinol does not readily exchange with the free quinol in the membrane (18Sato-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, 22Sato-Watanabe M. Mogi T. Miyoshi H. Anraku Y. Biochemistry. 1998; 37: 5356-5361Crossref PubMed Scopus (38) Google Scholar, 23Mogi T. Sato-Watanabe M. Miyoshi H. Orii Y. FEBS Lett. 1999; 457: 61-64Crossref PubMed Scopus (17) Google Scholar, 24Puustinen A. Verkhovsky M.I. Morgan J.E. Belevich N.P. Wikstrom M. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 1545-1548Crossref PubMed Scopus (69) Google Scholar). The ubiquinone bound at the QH-site functions as a cofactor, accepting two electrons from the quinol at the QL-site and passing the electrons on to heme b one at a time. Reduced (ferrous) heme b then transfers an electron to the heme o3/CuB active site, where O2 is reduced to 2H2O (25Matsuura K. Yoshioka S. Takahashi S. Ishimori K. Mogi T. Hori H. Morishima I. Biochemistry. 2004; 43: 2288-2296Crossref PubMed Scopus (3) Google Scholar). The ubiquinone bound at the QH-site of cyt bo3 forms a stable semiquinone when the protein is partially reduced (26Ingledew W.J. Ohnishi T. Salerno J.C. Eur. J. Biochem. 1995; 227: 903-908Crossref PubMed Scopus (70) Google Scholar, 27Sato-Watanabe M. Itoh S. Mogi T. Matsuura K. Miyoshi H. Anraku Y. FEBS Lett. 1995; 374: 265-269Crossref PubMed Scopus (64) Google Scholar). A combination of x-ray crystallography, site-directed mutagenesis, and pulsed EPR methods have been used to define the residues at the QH-site and details of the interactions between these residues and the bound semiquinone (3Lin M.T. Samoilova R.I. Gennis R.B. Dikanov S.A. J. Am. Chem. Soc. 2008; 130: 15768-15769Crossref PubMed Scopus (24) Google Scholar, 4Yap L.L. Samoilova R.I. Gennis R.B. Dikanov S.A. J. Biol. Chem. 2007; 282: 8777-8785Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 6Yap L.L. Samoilova R.I. Gennis R.B. Dikanov S.A. J. Biol. Chem. 2006; 281: 16879-16887Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 21Abramson 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 (358) Google Scholar, 28Hellwig P. Yano T. Ohnishi T. Gennis R.B. Biochemistry. 2002; 41: 10675-10679Crossref PubMed Scopus (33) Google Scholar, 29Grimaldi S. MacMillan F. Ostermann T. Ludwig B. Michel H. Prisner T. Biochemistry. 2001; 40: 1037-1043Crossref PubMed Scopus (37) Google Scholar, 30Grimaldi 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). Four polar residues have been implicated in binding to the quinol at the QH-site in cyt bo3: Arg-71, Asp-75, His-98, and Gln-101. Within the >400 sequences of quinol oxidases, Arg-71, Asp-75, and His-98 are totally conserved. Gln-101 is totally conserved in sequences from proteobacteria but is often replaced by a glutamic acid in the homologues in the Firmicutes, including the B. subtilis aa3-600 menaquinol oxidase. Pulsed EPR methods have revealed several salient features of the interactions between the residues at the QH-site of cyt bo3 and the SQ; 1) the hydrogen bonding to the SQ is highly asymmetric, with strong hydrogen bonds to carbonyl O-1 and weaker interactions at carbonyl O-4 side (6Yap L.L. Samoilova R.I. Gennis R.B. Dikanov S.A. J. Biol. Chem. 2006; 281: 16879-16887Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), 2) there is one strong hydrogen bond between the ϵ-nitrogen of Arg-71 and carbonyl O-1 of the SQ, resulting in a substantial transfer of unpaired electron spin to this nitrogen (3Lin M.T. Samoilova R.I. Gennis R.B. Dikanov S.A. J. Am. Chem. Soc. 2008; 130: 15768-15769Crossref PubMed Scopus (24) Google Scholar), 3) there is a strong hydrogen bond between Asp-75 and carbonyl O-1 of the SQ (4Yap L.L. Samoilova R.I. Gennis R.B. Dikanov S.A. J. Biol. Chem. 2007; 282: 8777-8785Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), 4) there is a weak interaction between His-98 and carbonyl O-4 of the SQ with a small amount of spin density found on the nitrogens of His-98, 5) there is a very weak interaction between carbonyl O-4 of the SQ and the side chain of Gln-101 (3Lin M.T. Samoilova R.I. Gennis R.B. Dikanov S.A. J. Am. Chem. Soc. 2008; 130: 15768-15769Crossref PubMed Scopus (24) Google Scholar), 6) the SQ in cyt bo3 is in the neutral, protonated state at pH 7.5 (6Yap L.L. Samoilova R.I. Gennis R.B. Dikanov S.A. J. Biol. Chem. 2006; 281: 16879-16887Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). In the current work it is demonstrated that the aa3-600 menaquinol oxidase from B. subtilis, isolated with the detergent dodecylmaltoside, contains 1 eq of bound menaquinone-7. Partial reduction of the enzyme results in formation of a SQ, analogous to the formation of the SQ formed at the QH-site in cyt bo3. In the B. subtilis aa3-600 menaquinol oxidase, the four residues at the putative QH-site are Arg-70, D74, His-94, and Glu-97. The SQ stabilized by the B. subtilis aa3-600 was examined using continuous-wave and pulsed EPR methods. The results show a distinctly different pattern of hydrogen bonding between the protein and SQ species in the menaquinol oxidase than that observed with the E. coli cyt bo3 ubiquinol oxidase. The qoxABCD operon, encoding the B. subtilis aa3-600 menaquinol oxidase, was cloned and expressed from plasmid pLala (Cmr) under the control of the glp promoter. Plasmid pLala replicates both in E. coli and B. subtilis. E. coli strains transformed with pLala vector were maintained on LB plates with 12 μg/ml chloramphenicol. A His6 tag was introduced at the C terminus of qoxB to faciliate protein purification by nickel-nitrilotriacetic acid. The isolated recombinant plasmid pLala was transformed into B. subtilis strain LUW143, lacking both the aa3-600 menaquinol oxidase and caa3-type cytochrome c oxidase (ΔqoxABCD::kan ΔctaCD::ble) (31Winstedt L. von Wachenfeldt C. J. Bacteriol. 2000; 182: 6557-6564Crossref PubMed Scopus (79) Google Scholar). Liquid cultures were inoculated with B. subtilis cells grown on LB plates containing appropriate antibiotics. Cells were grown in LB medium treated with 5 μg/ml chloramphenicol, 7.5 μg/ml neomycin, and 1.8 μg/ml zeomycin at 37 °C. Enzyme expression was induced by the addition of 20 mm glycerol. To isolate cell membranes, cell pellets were resuspended in buffer containing 50 mm K2HPO4 or 50 mm Tris plus 10 mm MgCl2 at pH 7.5 and disrupted at high pressure (100 p.s.i.) by using a microfluidizer (Microfluidics Corp., Worcester, MA). Cell debris was removed by brief centrifugation at 8000 × g. The supernatant was then subjected to centrifugation at 180,000 × g for at least 4 h to collect membranes. The isolated membranes were dispersed in 50 mm K2HPO4, pH 7.5, and homogenized with 1% dodecylmaltoside (Anatrace) by stirring at 4 °C. The solution was centrifuged at 180,000 × g for 1 h to remove insoluble fragments and then loaded onto a nickel-nitrilotriacetic acid column for purification. The column was initially equilibrated with 50 mm K2HPO4, 40 mm NaCl, 0.05% dodecylmaltoside, pH 7.5, for 3–5 column volumes. At least two incremental stepwise washes were performed with buffer containing up to 15 mm imidazole. Cytochrome aa3-600 was eluted with buffer containing 100 mm imidazole. The protein was dialyzed overnight against 50 mm K2HPO4, 0.05% dodecylmaltoside, pH 7.5, and concentrated to ∼200 μm. For the preparation of 15N-labeled protein sample, cells were grown in Spizizen minimal medium, where the nitrogen source was replaced with isotopically labeled 15NH4Cl (Cambridge Isotope, Andover, MA). Cyt aa3-600 oxidase had a turnover of 61 electrons s−1 at 25 °C with 2,3-dimethyl-1,4-naphthoquinone (DMN) in 50 mm Tris, 0.05%, pH 7.0. The respiratory activity was started by reducing 10 μm DMN in the presence of 200 μm NADH and excess amounts of purified diaphorase and by adding 0.05 μm of aa3-600 oxidase. The steady-state activity was monitored by oxidation of NADH at 340 nm. The autooxidation of DMN at concentrations above 10 μm prevented a study of oxidase activity dependence on substrate concentration. Among the various quinol-type electron donors, DMN was previously shown to have the highest enzyme activity (11Lemma E. Schägger H. Kröger A. Arch. Microbiol. 1993; 159: 574-578Crossref PubMed Scopus (22) Google Scholar). Quinone was extracted from the purified enzyme preparation with 3 ml of solvent containing methanol/petroleum ether (6:4, v/v) and repeated three times. The organic phase was combined and treated further after a procedure previously described (18Sato-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). The quinone was isolated by reverse-phase HPLC using a Varian Microsorb-MV 100–5 C18 column (4.6 mm × 25 cm) and a Waters HPLC system. Isoprenoid quinone structure was characterized by mass spectroscopy (Mass Spectrometry Laboratory, University of Illinois at Urbana-Champaign, Urbana, IL). To prepare the samples for EPR analysis, the purified protein (100–200 μm) was reduced anaerobically in the presence of a 3–5-fold excess of DMN and a 200-fold excess of NADH and rapidly frozen in the EPR tube. The continuous-wave EPR measurements were performed on an X-band Varian EPR-E122 spectrometer and a Q-band Bruker ELEXSYS 580 equipped with a separate Q-band microwave bridge and cavity operating at a 100 kHz modulation frequency. The pulsed EPR experiments were carried out using an X-band Bruker ELEXSYS E580 spectrometer equipped with Oxford CF 935 cryostats. Several types of experiments with different pulse sequences were employed with appropriate phase-cycling schemes to eliminate unwanted features from the experimental echo envelopes. Among these experiments were one- and two-dimensional three-pulse and four-pulse sequences, which are described in detail elsewhere (3Lin M.T. Samoilova R.I. Gennis R.B. Dikanov S.A. J. Am. Chem. Soc. 2008; 130: 15768-15769Crossref PubMed Scopus (24) Google Scholar). Spectral processing of three- and four-pulse ESEEM patterns was performed using Bruker WIN-EPR software, including subtraction of the relaxation decay (fitting by 3–6 degree polynomials), apodization (Hamming window), zero filling, and fast Fourier transformation. Pulsed ENDOR spectra of the radicals were obtained using Davies and Mims sequences with different pulse lengths. The specifics of these experiments are described in detail elsewhere (4Yap L.L. Samoilova R.I. Gennis R.B. Dikanov S.A. J. Biol. Chem. 2007; 282: 8777-8785Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 32Schweiger A. Jeschke G. Principles of Pulse Electron Paramagnetic Resonance. Oxford University Press, Oxford2001: 359-405Google Scholar). The preparation of his-tagged cytochrome aa3-600 was assayed for the presence of quinone. It was found that 1.25 eq of menaquinone-7 (Fig. 1) co-purifies with the enzyme, which is extracted from the membrane and purified using the detergent dodecylmaltoside. Fig. 2 shows X- and Q-band EPR spectra of the SQ in the wild type cyt aa3-600. The X-band spectrum displays a single pattern with a g value of 2.0047 ± 0.0001 and resolved hyperfine structure consisting of the four components with approximate relative intensities 1:3:3:1 and a splitting of ∼0.45–0.49 mT (or 12–13 MHz). The hyperfine structure resolution is better in the sample prepared in 2H2O. On the other hand, the uniform 15N labeling of the protein does not significantly influence the EPR line-shape of the SQ. This EPR feature is tentatively assigned to three equivalent nonexchangeable protons interacting with the unpaired electron. The Q-band spectrum, measured with about 3.5-times higher microwave frequency than the X-band, resolves the g-tensor anisotropy with components gxx = 2.00642 ± 0.00002, gyy = 2.00540 ± 0.00002, gzz = 2.00228 ± 0.00004. Additional hyperfine structure (also better resolved in the sample prepared in 2H2O) can be seen in the area around gyy; however, its complete resolution would require experiments at microwave frequencies ∼95 GHz or higher. The components of the g-tensor, determined from the Q-band spectra of cyt aa3-600 SQ prepared in H2O and 2H2O, are within the range previously reported for various SQs in model systems and in proteins (30Grimaldi 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, 33Lubtitz W. Feher G. Appl. Magn. Reson. 1999; 17: 1-48Crossref Scopus (130) Google Scholar, 34Veselov A.V. Osborne J.P. Gennis R.B. Scholes C.P. Biochemistry. 2000; 39: 3169-3175Crossref PubMed Scopus (30) Google Scholar). A Q-band spectrum with a similar shape was previously reported for the semiquinone intermediate stabilized in the membrane-bound subunit NarI of nitrate reductase (NarGHI) from E. coli (35Grimaldi S. Lanciano P. Bertrand P. Blasco F. Guigliarelli B. Biochemistry. 2005; 44: 1300-1308Crossref PubMed Scopus (25) Google Scholar). Powder-type ESEEM spectra, obtained with frozen protein solutions, do not usually show all of the 14N nuclei that are magnetically coupled with the SQ. This is due to the influence of the nuclear quadrupole interaction (3Lin M.T. Samoilova R.I. Gennis R.B. Dikanov S.A. J. Am. Chem. Soc. 2008; 130: 15768-15769Crossref PubMed Scopus (24) Google Scholar, 4Yap L.L. Samoilova R.I. Gennis R.B. Dikanov S.A. J. Biol. Chem. 2007; 282: 8777-8785Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 6Yap L.L. Samoilova R.I. Gennis R.B. Dikanov S.A. J. Biol. Chem. 2006; 281: 16879-16887Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 36Dikanov S.A. Holland J.T. Endeward B. Kolling D.R. Samoilova R.I. Prisner T.F. Crofts A.R. J. Biol. Chem. 2007; 282: 25831-25841Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). To observe all of the nitrogens that magnetically interact with the unpaired electron spin of the SQ, it is necessary to use 15N-labeled protein. The 15N nucleus is a spin ½ system and does not possess the nuclear quadrupole moment that affects the 14N ESEEM spectra. Therefore, we performed ESEEM experiments both with the wild type cyt aa3-600-containing natural abundance 14N (99.16%) and with uniformly 15N-labeled protein. Fig. 3 shows a representative 15N HYSCORE spectrum from a uniformly 15N-labeled sample, measured at the maximum of the SQ EPR line and displayed in contour (A) and three-dimensional-stacked (B) presentations. The 15N line-shape is centered around the sharp peak at a diagonal point (15νN,15νN) with a 15N Zeeman frequency 15νN ∼1.5 MHz. This peak is accompanied by extended shoulders with two weakly resolved maxima at (1.94, 1.08) MHz (1) and (1.73, 1.33) MHz (2) corresponding to couplings of 0.96 MHz and 0.4 MHz, respectively. The total length of the shoulders is ∼1.5 MHz along the antidiagonal, symmetrically around (15νN,15νN). This significantly exceeds typical values of the anisotropy for protein nitrogens interacting with a SQ (3Lin M.T. Samoilova R.I. Gennis R.B. Dikanov S.A. J. Am. Chem. Soc. 2008; 130: 15768-15769Crossref PubMed Scopus (24) Google Scholar, 36Dikanov S.A. Holland J.T. Endeward B. Kolling D.R. Samoilova R.I. Prisner T.F. Crofts A.R. J. Biol. Chem. 2007; 282: 25831-25841Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), suggesting that more than one nitrogen interacts with the SQ, accompanied by the transfer of unpaired spin density onto their nuclei. Major features of the 14N HYSCORE spectrum (Fig. 4, A and B) are the cross-peaks (1) correlating the frequencies at 3.5 and 4.5 MHz (±0.03 MHz). In addition, the spectrum resolves several other off-diagonal cross-features (2–4) of lower intensity, symmetrically located relative to the diagonal. The maxima of these cross-peaks are located at (3.0, 2.3) MHz (2), (3.0, 2.0) MHz (3), and (3.5, 2.0) MHz (4). Only the cross-peaks at (3.0, 2.3) MHz (2) possess a shape with well pronounced maxima. Cross-peaks 3 and 4 essentially have flat tops, indicating that they could be part of extended cross-features correlating transitions with significant orientation dependence. For this reason, the one-dimensional three-pulse ESEEM spectra show only two peaks at frequencies 3.5 and 4.4 MHz from transitions possessing a low orientation dependence in the HYSCORE spectra and do not resolve any other features (supplemental Fig. S1). The 3.5-, 3.0-, and 2.0-MHz frequencies are each made up of two different cross-peaks and cannot be assigned by assuming that all features are produced by the same nitrogen nucleus. A comparison of the frequencies of cross-peaks 1–4 shows that only 1 and 3 have a common frequency 3.5 MHz and, thus, could belong to the same nucleus. Cross-peaks 2 and 4 involve frequencies different from those of 1 and 3 and could be part of the extended cross-features correlating other transitions from either the same nucleus or from one or more different 14N nuclei. In summary, the 15N and 14N HYSCORE spectra indicate that the SQ in cyt aa3-600 interacts with at least two nitrogens from the protein environment. Besides the nitrogens, the HYSCORE spectra contain information about non-exchangeable and exchangeable protons interacting with the electron spin of the SQ. Fig. 5 shows the 1H HYSCORE spectra of the SQ in the cyt aa3-600 prepared in 1H2O (A and B) and 2H2O (C) buffer. Similar spectra for the SQ in the Qi-site of the cytochrome bc1 complex (37Dikanov S.A. Samoilova R.I. Kolling D.R. Holland J.T. Crofts A.R. J. Biol. Chem. 2004; 279: 15814-15823Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and QH-site of cytochrome bo3 (6Yap L.L. Samoilova R.I. Gennis R.B. Dikanov S.A. J. Biol. Chem. 2006; 281: 16879-16887Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) have been discussed previously. In addition to a diagonal peak with extended shoulders at the proton Zeeman frequency (νH∼14.75 MHz), the spectrum contains several pairs of resolved cross-features located symmetrically relative to the diagonal. They are designated 1, 2, 3, and 4. The cross-peaks labeled 1 demonstrate the largest hyperfine splitting, of the order ∼10 MHz. Cross-ridges 2 possess the most extended anisotropic contour, with the largest deviation from the diagonal indicating a significant anisotropic hyperfine component. Cross-peaks 3 and 4 are located in a similar area of the plot, close to each other and partially overlapping, as shown in Fig. 5B. Contours 1 and 3 are approximately normal to the diagonal, suggesting a smaller anisotropy. The contours of cross-peaks 4, located above those of cross-peaks 3, indicate an anisotropic hyperfine interaction intermediate between the couplings producing cross-peaks 3 and 2. Cross-peaks 2 and 4 (Fig. 5C) completely disappear in the HYSCORE spectra obtained under the same conditions using the sample with 2H2O, showing that they are produced by exchangeable protons. However, cross-peaks 1 and 3 as well as the diagonal peak, with its shoulders, still appear in the spectra obtained in 2H2O. The 1H/2H exchange does affect the intensity of cross-peaks 3, which indicates that these peaks result from the simultaneous contribution of exchangeable and non-exchangeable protons. Additional support for this conclusion was obtained from pulsed ENDOR spectra (see below). Quantitative analysis of the cross-peak contour line-shapes and simulations of the spectra, described in detail in Yap et al. (6Yap L.L. Samoilova R.I. Gennis R.B. Dikanov S.A. J. Biol. Chem. 2006; 281: 16879-16887Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and Dikanov et al. (37Dikanov S.A. Samoilova R.I. Kolling D.R. Holland J.T. Crofts A.R. J. Biol. Chem. 2004; 279: 15814-15823Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and the supplemental data (including supplemental Figs. S2 and S3 and Table S1) provides the isotropic (a) and anisotropic (T) components of the hyperfine tensors. These are obtained using an axial approximation for protons H1, H2, and H4 associated with cross-peaks 1, 2, and 4. The data are summarized in Table 1. Protons H2 and H4 are clearly exchangeable with the solvent. Cross-peaks 3 are produced by several non-exchangeable and weakly coupled exchangeable protons (see “Discussion”), and the parameters determined from this f" @default.
• W2036681907 created "2016-06-24" @default.
• W2036681907 creator A5008855880 @default.
• W2036681907 creator A5013262304 @default.
• W2036681907 creator A5019321812 @default.
• W2036681907 creator A5031834596 @default.
• W2036681907 creator A5048778417 @default.
• W2036681907 date "2010-06-01" @default.
• W2036681907 modified "2023-09-30" @default.
• W2036681907 title "Characterization of the Semiquinone Radical Stabilized by the Cytochrome aa3-600 Menaquinol Oxidase of Bacillus subtilis" @default.
• W2036681907 cites W1492994354 @default.
• W2036681907 cites W1516667549 @default.
• W2036681907 cites W1523705478 @default.
• W2036681907 cites W1524183389 @default.
• W2036681907 cites W1548394531 @default.
• W2036681907 cites W1550149310 @default.
• W2036681907 cites W1826802047 @default.
• W2036681907 cites W1898563571 @default.
• W2036681907 cites W1964647308 @default.
• W2036681907 cites W1965500486 @default.
• W2036681907 cites W1965510228 @default.
• W2036681907 cites W1966286780 @default.
• W2036681907 cites W1967943538 @default.
• W2036681907 cites W1971376700 @default.
• W2036681907 cites W1983312132 @default.
• W2036681907 cites W1983360419 @default.
• W2036681907 cites W1984370423 @default.
• W2036681907 cites W1986787486 @default.
• W2036681907 cites W1987765244 @default.
• W2036681907 cites W1988072095 @default.
• W2036681907 cites W1989160221 @default.
• W2036681907 cites W2007154856 @default.
• W2036681907 cites W2007176314 @default.
• W2036681907 cites W2010971001 @default.
• W2036681907 cites W2013436866 @default.
• W2036681907 cites W2018288289 @default.
• W2036681907 cites W2023151223 @default.
• W2036681907 cites W2024943035 @default.
• W2036681907 cites W2029667189 @default.
• W2036681907 cites W2032998875 @default.
• W2036681907 cites W2033920637 @default.
• W2036681907 cites W2040937743 @default.
• W2036681907 cites W2042197884 @default.
• W2036681907 cites W2042987383 @default.
• W2036681907 cites W2045404539 @default.
• W2036681907 cites W2046872945 @default.
• W2036681907 cites W2047182558 @default.
• W2036681907 cites W2049632955 @default.
• W2036681907 cites W2052721805 @default.
• W2036681907 cites W2062401684 @default.
• W2036681907 cites W2070900984 @default.
• W2036681907 cites W2080307773 @default.
• W2036681907 cites W2083832102 @default.
• W2036681907 cites W2093884442 @default.
• W2036681907 cites W2103070955 @default.
• W2036681907 cites W2110176312 @default.
• W2036681907 cites W2110719909 @default.
• W2036681907 cites W2124373056 @default.
• W2036681907 cites W2133623225 @default.
• W2036681907 cites W2152301430 @default.
• W2036681907 cites W2154158126 @default.
• W2036681907 cites W2162192550 @default.
• W2036681907 cites W2163749104 @default.
• W2036681907 cites W2164938252 @default.
• W2036681907 cites W2166258960 @default.
• W2036681907 cites W2172175523 @default.
• W2036681907 cites W3004630159 @default.
• W2036681907 cites W4238652547 @default.
• W2036681907 cites W4245255344 @default.
• W2036681907 cites W2047056237 @default.
• W2036681907 doi "https://doi.org/10.1074/jbc.m110.116186" @default.
• W2036681907 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2883873" @default.
• W2036681907 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/20351111" @default.
• W2036681907 hasPublicationYear "2010" @default.
• W2036681907 type Work @default.
• W2036681907 sameAs 2036681907 @default.
• W2036681907 citedByCount "25" @default.
• W2036681907 countsByYear W20366819072012 @default.
• W2036681907 countsByYear W20366819072013 @default.
• W2036681907 countsByYear W20366819072014 @default.
• W2036681907 countsByYear W20366819072015 @default.
• W2036681907 countsByYear W20366819072016 @default.
• W2036681907 countsByYear W20366819072017 @default.
• W2036681907 countsByYear W20366819072018 @default.
• W2036681907 countsByYear W20366819072019 @default.
• W2036681907 countsByYear W20366819072020 @default.
• W2036681907 countsByYear W20366819072021 @default.
• W2036681907 countsByYear W20366819072022 @default.
• W2036681907 countsByYear W20366819072023 @default.
• W2036681907 crossrefType "journal-article" @default.
• W2036681907 hasAuthorship W2036681907A5008855880 @default.
• W2036681907 hasAuthorship W2036681907A5013262304 @default.
• W2036681907 hasAuthorship W2036681907A5019321812 @default.
• W2036681907 hasAuthorship W2036681907A5031834596 @default.
• W2036681907 hasAuthorship W2036681907A5048778417 @default.
• W2036681907 hasBestOaLocation W20366819071 @default.
• W2036681907 hasConcept C123249941 @default.
• W2036681907 hasConcept C181199279 @default.

• next