• Journal of Microbiology
• /
• 제44권3호
• /
• pp.284-292
• /
• 2006

The cbb3 cytochrome c oxidase has the dual function as a terminal oxidase and oxygen sensor in the photosynthetic bacterium, Rhodobacter sphaeroides. The cbb3 oxidase forms a signal transduction pathway together with the PrrBA two-component system that controls photosynthesis gene expression in response to changes in oxygen tension in the environment. Under aerobic conditions the cbb3 oxidase generates an inhibitory signal, which shifts the equilibrium of PrrB kinase/phosphatase activities towards the phosphatase mode. Photosynthesis genes are thereby turned off under aerobic conditions. The catalytic subunit (CcoN) of the R. sphaeroides cbb3 oxidase contains five histidine residues (H2l4, B233, H303, H320, and H444) that are conserved in all CcoN subunits of the cbb3 oxidase, but not in the catalytic subunits of other members of copper-heme superfamily oxidases. H214A mutation of CcoN affected neither catalytic activity nor sensory (signaling) function of the cbb3 oxidase, whereas H320A mutation led to almost complete loss of both catalytic activity and sensory function of the cbb3 oxidase. H233V and H444A mutations brought about the partial loss of catalytic activity and sensory function of the cbb3 oxidase. Interestingly, the H303A mutant form of the cbb3 oxidase retains the catalytic function as a cytochrome c oxidase as compared to the wild-type oxidase, while it is defective in signaling function as an oxygen sensor. H303 appears to be implicated in either signal sensing or generation of the inhibitory signal to the PrrBA two-component system.

• BMB Reports
• /
• 제40권5호
• /
• pp.697-707
• /
• 2007

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.

• 생명과학회지
• /
• 제22권8호
• /
• pp.1009-1017
• /
• 2012

Rodobacter sphaeroides에서 orf282 유전자는 cbb3 terminal oxidase를 암호화하는 ccoNOQP 오페론과 혐기적 활성자인 FnrL을 암호화하는 fnrL 유전자 사이에 있으며, 아직은 기능이 잘 알려지지 않았다. orf282 유전자의 기능을 알기 위해 우리는 orf282의 일부를 삭제함으로써 유전자를 붕괴시켜 orf282-minus mutant를 제조하였다. 두개의 FnrL 결합 부위가 orf282의 upstream에 존재한다는 것이 밝혀져 있으며, orf282 유전자가 FnrL에 의해 양성적으로 조절된다는 것이 증명되었다. orf282 유전자는 B875와 B800-850 spectral complexes의 형성과 관련이 없다. orf282 mutant에서의 cbb3 oxidase 활성을 wild type와 비교해보면 orf282 유전자가 ccoNOQP 오페론의 조절과 cbb3 cytochrome c oxidase의 생합성과 무관하다는 것을 알 수 있다. orf282 mutant의 구조 유전자인 nifH와 조절유전자인 nifA의 프로모터 활성이 증가한 것은 orf282 유전자 산물이 nifH와 nifA의 발현에서 음성적 effector로 작용한다는 것을 시사한다.