Heme A is not essential for assembly of the subunits of cytochrome c oxidase of Rhodobacter sphaeroides.
The aa(3)-type cytochrome c oxidase of Rhodobacter sphaeroides, a proteobacterium of the alpha subgroup, is structurally similar to the core subunits of the terminal oxidase in the mitochondrial electron transport chain. Subunit I, the product of the coxI gene, normally binds two heme A molecules. A deletion of cox10, the gene for the farnesyltransferase required for heme A synthesis, did not prevent high level accumulation of subunit I in the cytoplasmic membrane. Thus, subunit I can be expressed and stably inserted into the cytoplasmic membrane in the absence of heme A. Aposubunit I was purified via affinity chromatography to a polyhistidine tag. Copurification of subunits II and III with aposubunit I indicated that assembly of the core oxidase complex occurred without the binding of heme A. In addition to formation of the apooxidase containing all three large structural proteins, CoxI-II and CoxI-III heterodimers were isolated from cox10 deletion strains harboring expression plasmids with coxI and coxII or with coxI and coxIII, respectively. This demonstrated that subunit assembly of the apoenzyme was not an inherently ordered or sequential process. Thus, multiple paths must be considered for understanding the assembly of this integral membrane metalloprotein complex.
Hiser
L
L
Department of Biochemistry, The University of Mississippi Medical Center, Jackson, Mississippi 39216, USA.
Hosler
J P
JP
D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D006418:Heme / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000502:physiology*; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012242:Rhodobacter sphaeroides / Q000201:enzymology*
C027728:heme a; D006418:Heme; D003576:Electron Transport Complex IV