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Investigation of cytochrome c dependent nitric oxide reductase (cNOR) from Paracoccus denitrificans
Abstracts
therefore analyzed the proteome of bacteria from acetate and pyruvate cultures in the exponential and stationary phase of growth. To our surprise H. modesticaldum expresses the entire pathway from acetate to butyrate when grown in the light on acetate + CO2, as well as in the stationary phase on pyruvate. References 1. L. Kimble, L. Mandelco, C. Woese, M. Madigan, Heliobacterium modesticaldum, sp. nov., a thermophilic heliobacterium of hot springs and volcanic soil, Arch Microbiol, 163 (1995) 259–267 2. W. Sattley, M. Madigan, W. Swingley, P. Cheung, K. Clocksin, A. Conrad, L. Dejesa, B. Honchak, D. Jung, L. Karbach, A. Kurdoglu, S. Lahiri, S. Mastrian, L. Page, H. Tayloe, Z. Wang, J. Raymond, M. Chen, R. Blankenship, J. Touchma, The genome of Heliobacterium modesticaldum, a phototrophic representative of the Firmicutes containing the simplest photosynthetic apparatus, J Bact, 190 (2008) 4687–4696. doi:10.1016/j.bbabio.2016.04.295
08.23 Investigation of cytochrome c dependent nitric oxide reductase (cNOR) from Paracoccus denitrificans Sinan Sabuncua, Josy Ter Beekb, Madeleine Stricklanda, Pia Ädelrothb, Frederic Melina, Petra Hellwiga a University of Strasbourg, UMR 7140, France b Stockholm University, Department of Biochemistry and Biophysics, Sweden E-mail address: [email protected] (S. Sabuncu) The Nitric oxide reductase (cNOR) has an important role in denitrification processes in bacteria and catalyzes the reaction of NO to N2O. It contains a low spin heme c, two b type hemes (low spin b, high spin b3) and one non-heme iron (FeB) [1]. This enzyme is related to the heme-copper oxidase superfamily of membrane proteins and it was suggested that cytochrome oxidases (COX) and NOR share a common ancestor [2]. A Ca2 + site was identified close to the hemes b and b3 which has a role in the electron transfer between these hemes and their conformation. In this study we work on wild type (WT) and mutant enzymes from P. denitrificans which are ligands of the Ca2 + site (E122A,Y74F and Y74S) [3]. The mid-point potentials of each heme was determined for wild type and mutant enzymes using UV/Vis potentiometric titrations and differential infrared spectroscopy was used to investigate changes in the protonation state of individual amino acids, secondary structure and environment of cofactors. Shifts in the mid-point potentials due to the single mutations at crucial residues are discussed. References 1. Y. Shiro, Structure and function of bacterial nitric oxide reductases: nitric oxide reductase, anaerobic enzymes, Biochimic. Biophys. Acta, 1817 (2012) 1907–1913 2. M. R. Cheesman, W. G. Zumft A. J., Thomson, The MCD and EPR of the heme centers of nitric oxide reductase from Pseudomonas stutzeri: evidence that the enzyme is structurally related to the heme-copper oxidases, Biochemistry-US, 37 (1998) 3994–4000 3. U. Flock, F. H. Thorndycroft, A. D. Matorin, D. J. Richardson, N. J. Watmough, P. Adelroth, Defining the proton entry point in the bacterial respiratory nitric-oxide reductase, J. Biol. Chem., 283 (2008) 3839–3845. doi:10.1016/j.bbabio.2016.04.296
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08.24 Cytochrome bd-type quinol oxidases: Structural variety even within the Gram-positive bacteria Junshi Sakamoto, Tsukasa Yoshida, Taichiro Hirose, Tomoichirou Kusumoto Kyushu Institute of Technology, Department of Bioscience and Bioinformatics, Kawazu 680-4, Iizuka, Fukuoka 820-8502, Japan E-mail address: [email protected] (J. Sakamoto) We have proposed two subfamilies of cytochrome bd quinol oxidase, S- and L-types [1]. They are different in the length of the Q loop, which is an important domain for quinol oxidation. We previously identified and purified cyt bd-type menaquinol oxidases from a thermophile Geobacillus thermodenitrificans K1041 and an amino acid-producing mesophile Corynebacterium glutamicum. These organisms are both Gram-positives and their cyts bd are both S-type, but the oxidases are much different in subunit sizes, optical property, and operon configuration. The wavelength of absorption peak due to the D-type heme is different as much as 10 nm. Two genes for subunits I and II cydAB are followed by genes for ABC transporter cydDC in the C. glutamicum genome, while by short orfs in the G. thermodenitrificans genome [2]. Recently, we solved the atomic structure of cyt bd from the thermophile [3]. This entirely novel structure itself and related biochemical analyses provide colorful insights into the molecular diversity of cyt bd. Crystal structures have been solved recently for complexes I through V and terminal oxidases including A-, B-, C-types of hemeCu oxidases etc. The cyt bd structure is the final piece to fill the bioenergetic jigsaw puzzle and useful to design novel bactericidal drugs. References 1. A.M. Arutyunyan, J. Sakamoto, Y. Kabashima, Y. Inadome. V.B. Borisov, Optical and magneto-optical activity of cytochrome bd from Geobacillus thermodenitrificans. Biochim. Biophys. Acta 1817 (2012) 2087–2094 2. J. Sakamoto, E. Koga, T. Mizuta, C. Sato, S. Noguchi, N. Sone, Gene structure and quinol oxidase activity of a cytochrome bd-type oxidase from Bacillus stearothermophilus. Biochim. Biophys. Acta 1411 (1999) 147–158 3. S. Safarian, H. Müller, C. Rajendran, J. Preu, J.D. Langer, S. Ovchinnikov, T. Hirose, T. Kusumoto, J. Sakamoto, H. Michel, The bd oxidases possess a unique protein structure and an unexpected heme group arrangement. Science (2016) in press. doi:10.1016/j.bbabio.2016.04.297
08.25 The aerobic respiratory NADH dehydrogenases in Escherichia coli Johannes Schimpf, Ina Schweizer, Thorsten Friedrich Institut für Biochemie, Albert-Ludwigs Universität, Freiburg, Germany E-mail address: [email protected] (J. Schimpf)
The E. coli aerobic respiratory chain basically consists of the NADH:ubiquinone oxidoreductase (complex I), the alternative NADH dehydrogenase (NDH-II), the succinate:ubiquinone oxidoreductase (complex II) and the terminal quinol oxidases bo3, bd-I and bd-II [1]. NADH from catabolic processes is used by complex I and NDH-II to reduce ubiquinone to ubiquinol. Subsequently, the electrons are transferred to the terminal oxidases to reduce oxygen to water.
therefore analyzed the proteome of bacteria from acetate and pyruvate cultures in the exponential and stationary phase of growth. To our surprise H. modesticaldum expresses the entire pathway from acetate to butyrate when grown in the light on acetate + CO2, as well as in the stationary phase on pyruvate. References 1. L. Kimble, L. Mandelco, C. Woese, M. Madigan, Heliobacterium modesticaldum, sp. nov., a thermophilic heliobacterium of hot springs and volcanic soil, Arch Microbiol, 163 (1995) 259–267 2. W. Sattley, M. Madigan, W. Swingley, P. Cheung, K. Clocksin, A. Conrad, L. Dejesa, B. Honchak, D. Jung, L. Karbach, A. Kurdoglu, S. Lahiri, S. Mastrian, L. Page, H. Tayloe, Z. Wang, J. Raymond, M. Chen, R. Blankenship, J. Touchma, The genome of Heliobacterium modesticaldum, a phototrophic representative of the Firmicutes containing the simplest photosynthetic apparatus, J Bact, 190 (2008) 4687–4696. doi:10.1016/j.bbabio.2016.04.295
08.23 Investigation of cytochrome c dependent nitric oxide reductase (cNOR) from Paracoccus denitrificans Sinan Sabuncua, Josy Ter Beekb, Madeleine Stricklanda, Pia Ädelrothb, Frederic Melina, Petra Hellwiga a University of Strasbourg, UMR 7140, France b Stockholm University, Department of Biochemistry and Biophysics, Sweden E-mail address: [email protected] (S. Sabuncu) The Nitric oxide reductase (cNOR) has an important role in denitrification processes in bacteria and catalyzes the reaction of NO to N2O. It contains a low spin heme c, two b type hemes (low spin b, high spin b3) and one non-heme iron (FeB) [1]. This enzyme is related to the heme-copper oxidase superfamily of membrane proteins and it was suggested that cytochrome oxidases (COX) and NOR share a common ancestor [2]. A Ca2 + site was identified close to the hemes b and b3 which has a role in the electron transfer between these hemes and their conformation. In this study we work on wild type (WT) and mutant enzymes from P. denitrificans which are ligands of the Ca2 + site (E122A,Y74F and Y74S) [3]. The mid-point potentials of each heme was determined for wild type and mutant enzymes using UV/Vis potentiometric titrations and differential infrared spectroscopy was used to investigate changes in the protonation state of individual amino acids, secondary structure and environment of cofactors. Shifts in the mid-point potentials due to the single mutations at crucial residues are discussed. References 1. Y. Shiro, Structure and function of bacterial nitric oxide reductases: nitric oxide reductase, anaerobic enzymes, Biochimic. Biophys. Acta, 1817 (2012) 1907–1913 2. M. R. Cheesman, W. G. Zumft A. J., Thomson, The MCD and EPR of the heme centers of nitric oxide reductase from Pseudomonas stutzeri: evidence that the enzyme is structurally related to the heme-copper oxidases, Biochemistry-US, 37 (1998) 3994–4000 3. U. Flock, F. H. Thorndycroft, A. D. Matorin, D. J. Richardson, N. J. Watmough, P. Adelroth, Defining the proton entry point in the bacterial respiratory nitric-oxide reductase, J. Biol. Chem., 283 (2008) 3839–3845. doi:10.1016/j.bbabio.2016.04.296
e91
08.24 Cytochrome bd-type quinol oxidases: Structural variety even within the Gram-positive bacteria Junshi Sakamoto, Tsukasa Yoshida, Taichiro Hirose, Tomoichirou Kusumoto Kyushu Institute of Technology, Department of Bioscience and Bioinformatics, Kawazu 680-4, Iizuka, Fukuoka 820-8502, Japan E-mail address: [email protected] (J. Sakamoto) We have proposed two subfamilies of cytochrome bd quinol oxidase, S- and L-types [1]. They are different in the length of the Q loop, which is an important domain for quinol oxidation. We previously identified and purified cyt bd-type menaquinol oxidases from a thermophile Geobacillus thermodenitrificans K1041 and an amino acid-producing mesophile Corynebacterium glutamicum. These organisms are both Gram-positives and their cyts bd are both S-type, but the oxidases are much different in subunit sizes, optical property, and operon configuration. The wavelength of absorption peak due to the D-type heme is different as much as 10 nm. Two genes for subunits I and II cydAB are followed by genes for ABC transporter cydDC in the C. glutamicum genome, while by short orfs in the G. thermodenitrificans genome [2]. Recently, we solved the atomic structure of cyt bd from the thermophile [3]. This entirely novel structure itself and related biochemical analyses provide colorful insights into the molecular diversity of cyt bd. Crystal structures have been solved recently for complexes I through V and terminal oxidases including A-, B-, C-types of hemeCu oxidases etc. The cyt bd structure is the final piece to fill the bioenergetic jigsaw puzzle and useful to design novel bactericidal drugs. References 1. A.M. Arutyunyan, J. Sakamoto, Y. Kabashima, Y. Inadome. V.B. Borisov, Optical and magneto-optical activity of cytochrome bd from Geobacillus thermodenitrificans. Biochim. Biophys. Acta 1817 (2012) 2087–2094 2. J. Sakamoto, E. Koga, T. Mizuta, C. Sato, S. Noguchi, N. Sone, Gene structure and quinol oxidase activity of a cytochrome bd-type oxidase from Bacillus stearothermophilus. Biochim. Biophys. Acta 1411 (1999) 147–158 3. S. Safarian, H. Müller, C. Rajendran, J. Preu, J.D. Langer, S. Ovchinnikov, T. Hirose, T. Kusumoto, J. Sakamoto, H. Michel, The bd oxidases possess a unique protein structure and an unexpected heme group arrangement. Science (2016) in press. doi:10.1016/j.bbabio.2016.04.297
08.25 The aerobic respiratory NADH dehydrogenases in Escherichia coli Johannes Schimpf, Ina Schweizer, Thorsten Friedrich Institut für Biochemie, Albert-Ludwigs Universität, Freiburg, Germany E-mail address: [email protected] (J. Schimpf)
The E. coli aerobic respiratory chain basically consists of the NADH:ubiquinone oxidoreductase (complex I), the alternative NADH dehydrogenase (NDH-II), the succinate:ubiquinone oxidoreductase (complex II) and the terminal quinol oxidases bo3, bd-I and bd-II [1]. NADH from catabolic processes is used by complex I and NDH-II to reduce ubiquinone to ubiquinol. Subsequently, the electrons are transferred to the terminal oxidases to reduce oxygen to water.