Abstract
Surf1p is a protein of the inner membrane of mitochondria that functions in the assembly of cytochrome-c oxidase. The specifics of the role of Surf1p have remained unresolved. Numerous mutations in human Surf1p lead to severe mitochondrial disease. A homolog of human Surf1p is encoded by the genome of the alpha-proteobacterium Rhodobacter sphaeroides, which synthesizes a mitochondrial-like aa(3)-type cytochrome-c oxidase. The gene for Surf1p was deleted from the genome of R. sphaeroides. The resulting aa(3)-type oxidase was purified and analyzed by biochemical methods plus optical and EPR spectroscopy. The oxidase that assembled in the absence of Surf1p was composed of three subpopulations with structurally distinct heme a(3)-Cu active sites. 50% of the oxidase lacked heme a(3), 10-15% contained heme a(3) but lacked Cu(BB), and 35-40% had a normal heme a(3) -Cu(B) active site with normal activity. Cu(A) assembly was unaffected. All of the oxidase contained low-spin heme a, but the environment of the heme a center was slightly altered in the 50% of the enzyme that lacked heme a(3). Introduction of a normal copy of the gene for Surf1p on an exogenous plasmid resulted in a single population of normally assembled, highly active enzyme. The data indicate that Surf1p plays a role in facilitating the insertion of heme a(3) into the active site of cytochrome-c oxidase. The results suggest that maturation of the heme a(3)-Cu(B) center is a step that limits the association of subunits I and II in the assembly of mitochondrial cytochrome oxidase.
Highlights
Cytochrome-c oxidase (CcO)1 functions as the terminal member of the respiratory electron transfer chain in mitochondria
Biochemical and Spectroscopic Characteristics of the aa3-type Cytochrome-c Oxidase That Assembles in the Absence of Surf1p—In the R. sphaeroides genome, surf1 is located immediately adjacent to the gene for subunit III of the aa3-type oxidase, as the first gene in what appears to be a four-gene operon
The CcO population that assembles in the absence of
Summary
Cytochrome-c oxidase (CcO) functions as the terminal member of the respiratory electron transfer chain in mitochondria. The genome of R. sphaeroides encodes homologs of five of these assembly proteins, which are present in yeast and human cells. PrrC is another copper-binding protein of R. sphaeroides with homology to eukaryotic Sco1 [10], the copper protein proposed to function in the assembly of CuA in mitochondria [6]. The predicted sizes of human (GenBankTM accession number NP_003163) and R. sphaeroides Surf1p (AY918925) are similar (300 versus 262 residues). Using EMBOSS-Align (European Bioinformatics Institute), the DNA-predicted amino acid sequence of R. sphaeroides Surf1p is 31% identical and 45% similar to human Surf1p. All of this homology is found in the extramembrane domains of the two proteins; the sequences of the transmembrane helices are not conserved. Surf1p of R. sphaeroides is no less similar to human Surf1p than is Shy1p of
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