A cb-type cytochrome-c oxidase terminates the respiratory chain in Helicobacter pylori

A Helicobacter pylori membrane fraction oxidized yeast and equine cytochrome c, and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). When ascorbate was used as reductant, the V-max and apparent K-m values were 612 nmol electron min(-1) (mg protein)(-1) and 14 mu M for yeast, and 419 nmol electron min(-1) (mg protein)(-1) and 19 mu M for equine cytochrome c, respectively. For TMPD oxidation, the V-max and K-m values were 640 nmol electron min(-1) (mg protein)(-1) and 182 mu M, respectively. These oxidase activities showed a high affinity for oxygen. Inhibition of both cytochrome-c and TMPD oxidase activities by 50% was caused by about 4 mu M cyanide and about 0.5 mM azide. Redox difference spectra of the membrane solubilized with Triton X-100 showed b- or c-type cytochromes but not aa(3)-type cytochromes. c-type and a part of some b-type cytochromes were reduced with ascorbate plus TMPD, A CO difference spectrum revealed that protohaem, but not an aa(3)-type cytochrome, may be interacting with CO/oxygen. Only protohaem was detected in the haem fraction extracted from the membrane. Three polypeptides (60, 38 and 29 kDa) were found to be bearing haem c after SDS-PACE of the membrane. From these results, it was suggested that the cbb(3)-type cytochrome-c oxidase, having a haem-copper binuclear centre like the cytochrome aa(3)-type oxidase, but differing in a few other properties, functions as a terminal oxidase in the respiratory chain of H. pylori.