Site-directed mutagenesis of five conserved residues of subunit I of the cytochrome cbb(3) oxidase in Rhodobacter capsulatus
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Dosyalar
Tarih
2007
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Springer Singapore Pte Ltd
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Cytochrome ebb.; 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 TNBZ 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.
Açıklama
Anahtar Kelimeler
Cytochrome cbb(3)-type Oxidase Activity, Respiration and Site-directed Mutagenesis, Rhodobacter Capsulatus
Kaynak
Journal Of Biochemistry And Molecular Biology
WoS Q Değeri
Q3
Scopus Q Değeri
N/A
Cilt
40
Sayı
5
