Characterization of a cb-type cytochrome c oxidase from Helicobacter pylori.
Helicobacter pylori is a microaerophilic Gram-negative spiral bacterium residing in human stomach. A cb-type cytochrome c oxidase that terminates the respiratory chain was purified to near homogeneity by solubilizing the membranes with Triton X-100 and applying anion exchange, Cu-chelating, and gel filtration chromatographies. Redox- and CO-difference spectra and pyridine ferrohaemochrome analysis showed the enzyme to contain three haems C, one low-spin protohaem, and one high-spin protohaem that probably forms a dioxygen-reducing bimetalic center with a copper atom. The enzyme actively oxidizes soluble cytochrome c from this bacterium (TNmax of about 250 s-1) with a Km of 0.9 microM. Yeast cytochrome c and N,N,N',N'-tetramethyl p-phenylenediamine (TMPD) are also oxidized at similar maximal velocities with larger Km's. Oxygen pulse experiments on resting cells in the presence of ascorbate plus TMPD or L-lactate indicated that this sole terminal oxidase pumps H+, although the H+ pumping activity by proteoliposomes reconstituted from the enzyme and P-lipids was low. Two main bands with haem C at 58 and 26 kDa were observed upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and succeeding protein and haem staining. Sequencing of the operon encoding the subunits of the enzyme revealed the presence of ccoNOQP. N-terminal analysis of the 58 kDa band showed 15 or 13 amino acids coinciding with the amino acid sequences deduced from the DNA of ccoN and ccoO. CcoN, the largest subunit bearing two protohaems and copper, and ccoO, a mono-haem cytochrome subunit form a protein complex with an apparent molecular mass of 58 kDa, even in the presence of sodium dodecyl sulfate. The 26 kDa band is tentatively assumed to be ccoP with two haems C.
Tsukita
S
S
Department of Biochemical System Engineering, Kyushu Institute of Technology, Iizuka, Fukuoka, 820-8502, Japan.
Koyanagi
S
S
Nagata
K
K
Koizuka
H
H
Akashi
H
H
Shimoyama
T
T
Tamura
T
T
Sone
N
N
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology; D002384:Catalysis; D003001:Cloning, Molecular; D003574:Cytochrome c Group / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D016480:Helicobacter pylori / Q000201:enzymology*; D006859:Hydrogen / Q000378:metabolism; D008969:Molecular Sequence Data; D009876:Operon; D010802:Phylogeny
D003574:Cytochrome c Group; C083951:cytochrome c553; D006859:Hydrogen; C022302:cytochrome C-552; D003576:Electron Transport Complex IV