Two conserved non-canonical histidines are essential for activity of the cbb (3)-type oxidase in Rhodobacter capsulatus: non-canonical histidines are essential for cbb (3)-type oxidase activity in R. capsulatus.
Cytochrome cbb (3) oxidase, a member of the heme-copper oxidase superfamily, catalyses the reduction of oxygen to water and generates a proton gradient. Cytochrome c oxidases are characterized by a catalytic subunit (subunit I) containing two hemes and one copper ion ligated by six invariant histidine residues, which are diagnostic of heme-copper oxidases in all type of the heme-copper oxidase superfamily. Alignments of the amino acid sequences of subunit I (FixN or CcoN) of the cbb (3)-type oxidases show that catalytic subunit also contains six non-canonical histidine residues that are conserved in all CcoN subunits of the cbb (3) oxidase, but not the catalytic subunits of other members of heme-copper oxidases superfamily. The function of these six CcoN-specific conserved histidines of cbb (3)-type oxidase in R. capsulatus is unknown. To analyze the contribution of the two invariant histidines of CcoN, H300 and H394, in activity and assembly of the Rhodobacter capsulatus cbb (3)-type oxidase, they were substituted for valine and alanine, respectively by site-directed mutagenesis. H300V and H394A mutations were analyzed with respect to their activity and assembly. It was found that H394A mutation led to a defect in the assembly of both CcoP and CcoO in the membrane, which results in almost complete loss of activity and that although the H300V mutant is normally assembled in the membrane and retain their stability, its catalytic activity is significantly reduced when compared with wild-type oxidase.
Oztürk
Mehmet
M
Faculty of Literature and Science, Biology Department, Abant Izzet Baysal University, Bolu, Turkiye.
Mandaci
Sevnur
S
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D017124:Conserved Sequence; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000737:chemistry; D006639:Histidine / Q000032:analysis* / Q000235:genetics; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D016354:Rhodobacter capsulatus / Q000201:enzymology* / Q000235:genetics
D006418:Heme; D006639:Histidine; C414443:cbb3 oxidase; D003576:Electron Transport Complex IV