Abstract
Menaquinol oxidase isolated from the membrane of Bacillus subtilis W23 was found to consist of four polypeptides (QoxA, B, C, and D) that were predicted by the sequence of the qox operon of B. subtilis 168 (Santana et al. 1992). The preparation contained 7 mol cytochrome aa 3 per g protein, which corresponds to 2mol heme A per mol enzyme of 144 kDa molecular mass. Respiration with dimethylnaphthoquinol catalyzed by the enzyme was ten times faster than that with menadiol. Activities with more electropositive quinols were negligible. The activity of the enzyme was inhibited by equimolar amounts of HQNO, while antimycin, myxothiazol, and stigmatellin were more than tenfold less effective. When cells of both strains of B. subtilis (W23 and 168) were grown with glucose, quinol respiration was an order of magnitude more active than respiration with N,N,N′,N′-tetramethyl-1,4-phenylenediamine plus ascorbate. Surprisingly, the same result was obtained with mutant strains lacking qoxB. As cytochromes a and d were virtually absent, a second quinol oxidase, possibly of the cytochrome o-type, was apparently formed by the mutants.
Similar content being viewed by others
Abbreviations
- cat :
-
Chloramphenicol resistance gene
- cta :
-
Cytochrome oxidase genes
- DMN :
-
2,3-Dimethyl-1,4-naphthoquinone
- DMNH 2 :
-
Reduced DMN
- HQNO :
-
2-(n-Heptyl)-4-hydroxyquinoline-N-oxide
- qox :
-
Quinol oxidase genes
- TMPD :
-
N,N,N′,N′-tetramethyl-1,4-phenylenediamine
References
Anagnostopoulos C, Spizizen J (1961) Requirements for transformation in Bacillus subtilis. J Bacteriol 81:741–746
Bergsma J, Meihuizen KE, Oeveren WV van, Konings WN (1982) Restoration of NADH oxidation with menaquinones and menaquinone analogues in membrane vesicles from the manquinone-deficient Bacillus subtilis aroD. Eur J Biochem 125: 651–657
Bernhard K, Schrempf H, Goebel W (1978) Bacteriocin and antibiotic resistance plasmids in Bacillus cereus and Bacillus subtilis. J Bacteriol 133:897–903
Bode C, Goebell H, Stähler E (1968) Zur Eliminierung von Trübungsfehlern bei der Eiweißbestimmung mit der Biuretmethode. Z Klin Chem Klin Biochem 6:418–422
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Chepuri V, Lemieux L, Au DCT, Gennis RB (1990) The sequence of the cyo operon indicates substantial structural similarities between the cytochrome o ubiquinol oxidase of Escherichia coli and the aa 3 -type family of cytochrome c oxidases. J Biol Chem 265:11185–11192
Dower WJ, Miller JF, Ragsdale CW (1988) High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 16:6127–6145
Dunbar BS, Schwoebel ED (1990) Preparation of polyclonal antibodies. Methods Enzymol 182:663–670
Fukaya M, Tayama K, Tamaki T, Ebisuya H, Okumura H, Kawamura Y, Horingouchi S, Beppu T (1993) Characterization of a cytochrome a 1 that functions as a ubiquinol oxidase in Acetobacter aceti. J Bacteriol 175:4307–4314
Garcia-Horsman JA, Barquera B, Rumbley J, Ma J, Gennis RB (1994) The superfamily of heme-copper respiratory oxidases. J Bacteriol 176:5587–5600
Hawkes R, Niday E, Gordon J (1982) A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem 119:142–147
James WS, Gibson F, Taroni P, Poole RK (1989) The cytochrome oxidases of Bacillus subtilis: mapping of a gene affecting cytochrome aa 3 and its replacement by cytochrome o in a mutant strain. FEMS Microbiol Lett 58:277–282
Kessler C, Höltke HJ, Seibi R, Burg J, Mühlegger K (1990) Nonradioactive labeling and detection of nucleic acids. A novel DNA labeling and detection system based on digoxigenin: antidigoxigenin ELISA principle (digoxigenin system). Biol Chem Hoppe Seyler 371:917–927
Klein C, Kaletta C, Schnell N, Entian KD (1992) Analysis of genes involved in biosynthesis of the lantibiotic subtilin. Appl Environ Microbiol 58:132–142
Kyhse-Andersen J (1984) Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods 10:203–209
Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lauraeus M, Wikström M (1993) The terminal quinol oxidases of Bacillus subtilis have different energy conservation properties. J Biol Chem 268:11470–11473
Lauraeus M, Haltia T, Saraste M, Wikström M (1991) Bacillus subtilis expresses two kinds of haem-A-containing terminal oxidases. Eur J Biochem 197:699–705
Lauraeus M, Morgan JE, Wikström M (1993) Peroxy and ferryl intermediates of the quinol-oxidizing cytochrome aa 3 from Bacillus subtilis. Biochem 32:2664–2670
Lemma E, Unden G, Kröger A (1990) Menaquinone is an obligatory component of the chain catalyzing succinate respiration in Bacillus subtilis. Arch Microbiol 155:62–67
Lemma E, Hägerhäll C, Geisler V, Brandt U, Jagow G von, Kröger A (1991) The reactivity of the Bacillus subtilis succinate dehydrogenase complex with quinones. Biochim Biophys Acta 1059:281–285
Lemma E, Schägger H, Kröger A (1993) The menaquinol oxidase of Bacillus subtilis W23. Arch Microbiol 159:574–578
Link TA, Haase U, Brandt U, Jagow G von (1993) What information do inhibitors provide about the structure of the hydroquinone oxidation site of ubihydroquinone: cytochrome c oxidoreductase? J Bioenerg Biomembr 25:221–232
Oost J van der, Wachenfeld C von, Hederstedt L, Saraste M (1991) Bacillus subtilis cytochrome oxidase mutants: biochemical analysis and genetic evidence for two aa 3 -type oxidases. Mol Microbiol 5:2063–2072
Powers L, Lauraeus M, Reddy KS, Chance B, Wikström M (1994) Structure of the binuclear heme iron-copper site in the quinoloxidizing cytochrome aa 3 from Bacillus subtilis. Biochim Biophys Acta 1183:504–512
Richter OMH, Tao J, Turba A, Ludwig B (1994) A cytochrome ba 3 functions as a quinol oxidase in Paracoccus denitrificans. J Biol Chem 269:23079–23086
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467
Santana M, Kunst F, Hullo MF, Rapoport G, Danchin A, Glaser P (1992) Molecular cloning, sequencing, and physiological characterization of the qox operon from Bacillus subtilis encoding the aa 3 -600 quinol oxidase. J Biol Chem 267:10225–10231
Saraste M, Metso T, Nakari T, Jalli T, Lauraeus M, Oost J van der (1991) The Bacillus subtilis cytochrome-c oxidase. Variations on a conserved protein theme. Eur J Biochem 195:517–525
Schägger H, Jagow G von (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379
Schnorf U (1966) PhD thesis, no. 3871. Swiss Federal Institute of Technology, Zurich
Vrij W de, Burg B van den, Konings WN (1987) Spectral and potentiometric analysis of cytochromes from Bacillus subtilis. Eur J Biochem 166:589–595
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Lemma, E., Simon, J., Schägger, H. et al. Properties of the menaquinol oxidase (Qox) and of qox deletion mutants of Bacillus subtilis . Arch. Microbiol. 163, 432–438 (1995). https://doi.org/10.1007/BF00272132
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00272132