Two conserved non-canonical histidines are essential for activity of the cbb
3-type oxidase in Rhodobacter capsulatus |
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Authors: | Mehmet Öztürk Sevnur Mandaci |
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Institution: | (1) Faculty of Literature and Science, Biology Department, Abant İzzet Baysal University, Bolu, 14280, Turkiye;(2) Genetic Engineering and Biotechnology Institute, TUBITAK-MRC, Gebze Campus, P.O. Box 21, Gebze, 41470, Kocaeli, Turkiye |
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Abstract: | 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. |
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Keywords: | Cytochrome cbb 3-type oxidase activity Respiration Rhodobacter capsulatus and site-directed mutagenesis |
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