Purification of Helicobacter pylori NCTC 11637 cytochrome bc1 and respiration with D-proline as a substrate.
Helicobacter pylori is a microaerophilic bacterium associated with gastric inflammation and peptic ulcers. Knowledge of how pathogenic organisms produce energy is important from a therapeutic point of view. We found d-amino acid dehydrogenase-mediated electron transport from d-proline or d-alanine to oxygen via the respiratory chain in H. pylori. Coupling of the electron transport to ATP synthesis was confirmed by using uncoupler reagents. We reconstituted the electron transport chain to demonstrate the electron flow from the d-amino acids to oxygen using the recombinant cytochrome bc(1) complex, cytochrome c-553, and the terminal oxidase cytochrome cbb(3) complex. Upon addition of the recombinant d-amino acid dehydrogenase and d-proline or d-alanine to the reconstituted electron transport system, reduction of cytochrome cbb(3) and oxygen consumption was revealed spectrophotometrically and polarographically, respectively. Among the constituents of H. pylori's electron transport chain, only the cytochrome bc(1) complex had been remained unpurified. Therefore, we cloned and sequenced the H. pylori NCTC 11637 cytochrome bc(1) gene clusters encoding Rieske Fe-S protein, cytochrome b, and cytochrome c(1), with calculated molecular masses of 18 kDa, 47 kDa, and 32 kDa, respectively, and purified the recombinant monomeric protein complex with a molecular mass of 110 kDa by gel filtration. The absorption spectrum of the recombinant cytochrome bc(1) complex showed an alpha peak at 561 nm with a shoulder at 552 nm.
Tanigawa
Minoru
M
Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University, 1-8-14 Kanda-Surugadai, Chiyoda Ward, Tokyo 101-8308, Japan.
Shinohara
Tomomitsu
T
Nishimura
Katsushi
K
Nagata
Kumiko
K
Ishizuka
Morio
M
Nagata
Yoko
Y
D000409:Alanine / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D014450:Electron Transport Complex III / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D016480:Helicobacter pylori / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D011048:Polarography / Q000379:methods; D011392:Proline / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013053:Spectrophotometry / Q000379:methods
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011392:Proline; D014450:Electron Transport Complex III; D000409:Alanine; D010100:Oxygen