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Site-directed Mutagenesis of Five Conserved Residues of Subunit I of the Cytochrome cbb3 Oxidase in Rhodobacter capsulatus

  • Ozturk, Mehmet (Abant zzet Baysal University, Faculty of Literature and Science, Biology Department) ;
  • Gurel, Ekrem (Abant zzet Baysal University, Faculty of Literature and Science, Biology Department) ;
  • Watmough, Nicholas J. (Center for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia) ;
  • Mandaci, Sevnur (Genetic Engineering and Biotechnology Institute, TUBITAK MRC)
  • Published : 2007.09.30
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Abstract

Cytochrome $cbb_3$ oxidase is a member of the heme-copper oxidase superfamily that catalyses the reduction of molecular oxygen to the water and conserves the liberated energy in the form of a proton gradient. Comparison of the amino acid sequences of subunit I from different classes of heme-copper oxidases showed that transmembrane helix VIII and the loop between transmembrane helices IX and X contain five highly conserved polar residues; Ser333, Ser340, Thr350, Asn390 and Thr394. To determine the relationship between these conserved amino acids and the activity and assembly of the $cbb_3$ oxidase in Rhodobacter capsulatus, each of these five conserved amino acids was substituted for alanine by site-directed mutagenesis. The effects of these mutations on catalytic activity were determined using a NADI plate assay and by measurements of the rate of oxygen consumption. The consequence of these mutations for the structural integrity of the $cbb_3$ oxidase was determined by SDS-PAGE analysis of chromatophore membranes followed by TMBZ staining. The results indicate that the Asn390Ala mutation led to a complete loss of enzyme activity and that the Ser333Ala mutation decreased the activity significantly. The remaining mutants cause a partial loss of catalytic activity. All of the mutant enzymes, except Asn390Ala, were apparently correctly assembled and stable in the membrane of the R. capsulatus.

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References

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