Deletion of the gene for subunit III leads to defective assembly of bacterial cytochrome oxidase.
COIII is one of the major subunits in the mitochondrial and a bacterial cytochrome c oxidase, cytochrome aa3. It does not contain any of the enzyme's redox-active metal centres and can be removed from the enzyme without major changes in its established functions. We have deleted the COIII gene from Paracoccus denitrificans. The mutant still expresses spectroscopically detectable enzyme almost as the wild-type, but its cytochrome c oxidase activity is much lower. From 50 to 80% of cytochrome a is reduced and its absorption maximum is 2-3 nm blue-shifted. The EPR signal of ferric cytochrome a is heterogeneous indicating the presence of multiple cytochrome a species. Proteolysis of the membrane-bound oxidase shows new cleavage sites both in COI and COII. DEAE-chromatography of solubilized enzyme yields fractions that contain a COI + COII complex and in addition haem-binding, free COI as well as free COII. The mutant phenotype can be complemented by introducing the COIII gene back to cells in a plasmid vector. We conclude that cytochrome oxidase assembles inefficiently in the absence of COIII and that this subunit may facilitate a late step in the assembly. The different oxidase species in the mutant represent either accumulating intermediates of the assembly pathway or dissociation products of a labile COI + COII complex and its conformational variants.
Haltia
T
T
Department of Medical Chemistry, University of Helsinki, Finland.
Finel
M
M
Harms
N
N
Nakari
T
T
Raitio
M
M
Wikström
M
M
Saraste
M
M
D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D002872:Chromosome Deletion; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006868:Hydrolysis; D009154:Mutation; D010231:Paracoccus denitrificans / Q000201:enzymology / Q000235:genetics*; D010447:Peptide Hydrolases; D010641:Phenotype; D010957:Plasmids; D012995:Solubility; D013057:Spectrum Analysis
D003576:Electron Transport Complex IV; D010447:Peptide Hydrolases