Molecular Biology Reports

, Volume 38, Issue 5, pp 3319–3326 | Cite as

Mutagenesis of tyrosine residues within helix VII in subunit I of the cytochrome cbb 3 oxidase from Rhodobacter capsulatus



The cbb 3-type oxidases are members of the heme-copper oxidase superfamily, distant by sequence comparisons, but sharing common functional characteristics. The cbb 3 oxidases are missing an active-site tyrosine residue that is absolutely conserved in all A and B-type heme-copper oxidases. This tyrosine is known to play a critical role in the catalytic mechanisms of A and B-type oxidases. The absence of this tyrosine in the cbb 3 oxidases raises the possibility that the cbb 3 oxidases utilize a different catalytic mechanism from that of the other members of the superfamily, or have this conserved residue in different helices. Recently sequence comparisons indicate that, a tyrosine residues that might be analogous to the active-site tyrosine in other oxidases are present in the cbb 3 oxidases but these tyrosines originates from a different transmembrane helix within the protein. In this research, three conserved tyrosine residues, Y294, Y308 and Y318, in helix VII were substituted for phenylalanine. Y318F mutant in the Rhodobacter capsulatus oxidase resulted in a fully assembled enzyme with nativelike structure and activity, but Y294F mutant is not assembled and have a catalytic activity. On the other hand, Y308F mutant is fully assembled enzyme with nativelike structure, but lacking catalytic activity. This result indicates that Y308 should be crucial in catalytic activity of the cbb 3 oxidase of R. capsulatus. These findings support the assumption that all of the heme-copper oxidases utilize the same catalytic mechanism and provide a residue originates from different places within the primary sequence for different members of the same superfamily.


Cytochrome cbb3-type oxidase Proton pumping channels Tyrosine–histidine cross-link Site-directed mutagenesis 



We thank Dr. Daldal for providing the strains MT1131 and GK32; and plasmids, HB101/pOX15. This work was partially supported by The Scientific and Technological Research Council of Turkey (TÜBİTAK) through TBAG-107T519 (to M.Ö.) and BİDEP (to M.Ö.); and European Molecular Biology Organization (EMBO) for short term fellowship (to M.Ö.).


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© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  1. 1.Department of Biology, Faculty of Literature and ScienceAbant İzzet Baysal UniversityBoluTurkey
  2. 2.Biological ScienceUniversity of East AngliaNorwichUK

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