Site-directed mutagenesis of five conserved residues of subunit i of the cytochrome cbb3 oxidase in Rhodobacter capsulatus.
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.
Ozturk
Mehmet
M
Abant zzet Baysal University, Faculty of Literature and Science, Biology Department, 14280, Bolu, Turkey.
Gurel
Ekrem
E
Watmough
Nicholas J
NJ
Mandaci
Sevnur
S
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D045304:Cytochromes c / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000379:methods; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013912:Threonine / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D001426:Bacterial Proteins; D021122:Protein Subunits; D013912:Threonine; D006418:Heme; D012694:Serine; D001216:Asparagine; D045304:Cytochromes c; C414443:cbb3 oxidase; D003576:Electron Transport Complex IV