Heme A biosynthetic pathway. The conversion of heme B to heme O is catalyzed by Cox10p, a farnesyl transferase. Arh1p (adrenodoxin reductase), Yah1p (adrenodoxin), and Cox15p jointly catalyze the hydroxylation of the methyl group at C8 of heme O producing an intermediate whose conversion to heme A is catalyzed by a still unidenti fi ed dehydrogenase. nHFe refers to the non-heme iron of adrenodoxin and hFe to the heme iron of Cox15p. 

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... also displayed by cox20 mutants. In this case, however, the processing block is not due to a lack of export of the amino terminal presequence or a defect in the protease. Instead the COX20 product appears to function as a subunit 2-speci fi c chaperone (Hell et al., 2000). This inner membrane protein interacts with the precursor to form a complex that is recognized as the proper substrate by the protease (Hell et al., 2000). Biogenesis of the membrane forms of subunits 1 and 3 is less well understood. Earlier studies indicated that insertion of the two proteins into the inner membrane requires the help of Oxa1p (Hell et al., 1998). This is also supported by more recent evidence of a physical interaction between Oxa1p and newly synthesized but unassembled subunits 1 and 3 (Hell et al., 2001). Even though information about these hydrophobic components is scant, it is not unreasonable to think that their interaction with each other and with subunit 2 to form a core complex can occur independent of the subunits synthesized in the cytosol. This is already implicit from evidence of bacterial cytochrome oxidases that have the same core structure as the mitochondrial enzyme and assemble into a stable complex even though the other subunits are absent. The existence of contact interfaces between each of the three core subunits also makes it unlikely that the subunits synthesized in the cytosol contribute signi fi cantly to the stability of the core complex. Based on the structure of the bovine COX (Tsukihara et al., 1996), only subunits Vb (yeast subunit 4), which makes contact with both subunits I and III, and subunit VIb (yeast subunit 6b), which contacts subunits II and III, could in fl uence the stability of the core complex (Table 1). The core subunits have the hallmarks of a protective shield that surrounds and caps a good portion of the exposed surfaces of the enzymatic core (Fig. 1). This is not to say, however, that there needs to be an obligatory order of subunit interaction or that interactions cannot occur between some subunits prior to formation of the core. For example, an intermediate of subunits I and IV has been detected in human mitochondria (Nijtmans et al., 1998). Heme A is a unique heme compound present only in cytochrome oxidase. The two heme A groups of cytochrome oxidase are non-covalently bound to subunit 1; they contribute to the low-spin heme of cytochrome a and the high-spin heme of cytochrome a 3 (Saraste, 1990). Heme A differs from protoheme (heme B) at carbons C2 and C8 of the porphyrin ring. It has a farnesyl instead of a vinyl group at C2, and a formyl instead of a methyl group at C8 (Caughey et al., 1975). The fi rst step in heme A biosynthesis is a farnesylation of the vinyl at C2 of protoheme (Saiki et al., 1993). In yeast, this reaction is catalyzed by a farnesyl transferase encoded by the COX10 to produce heme O (Tzagoloff et al., 1993). Heme O can function as a prosthetic group in some bacterial (Puustinen and Wikstrom, 1991) but not mitochondrial cytochrome oxidases. The further conversion of heme O to heme A probably involves a monooxy- genase-catalyzed hydroxylation of the methyl group at carbon position 8. The resultant alcohol would then be further oxidized to the aldehyde by a dehydrogenase. The cta A gene of Bacillus subtilis has been shown to be required for the conversion of heme O to heme A (Svensson et al., 1996). Escherchia coli , which normally has only heme O, is able to synthesize heme A when transformed with cta A (Svensson et al., 1993). Puri fi ed CtaA protein has both protoheme and heme A associated with it, suggesting that it is likely to be a heme-dependent monooxygenase (Svensson and Hederstedt, 1994; Svensson et al., 1996). Remarkably, CtaA has no homology to other known P450 cytochromes. We recently proposed that COX15 is the yeast homolog of cta A (Barros et al., 2001). Cox15p exhibits some sequence similarity to the bacterial protein. More signi fi cantly, cox15 mutants are COX-de fi cient and have no heme A, although they have low levels of heme O (Barros et al., 2001). This phenotype is different from cox10 mutants that lack both heme A and heme O (Tzagoloff et al., 1993). In Schizosaccharomyces pombe COX15 is fused to YAH1 , the structural gene for mitochondrial adrenodoxin (Barros and Nobrega, 1999). Yah1p, an essential protein in S. cerevisiae , has been shown to function in the assembly of iron- sulfur clusters (Lange et al., 2000). A fusion of the S. cere- visiae COX15 and YAH1 genes, when introduced in single copy into chromosomal DNA, complements the respiratory defect of a cox15 null mutant and the lethality of a yah1 mutant, excluding any effect of the combined presence of the two proteins in a single polypeptide on their respective activities (Barros et al., 2001). These observations suggest that Cox15p in conjunction with Yah1p and adrenodoxin reductase encoded by ARH1 (Manzella et al., 1998) function as a three component monooxygenase (Fig. 3). Mitochondrial cytochrome oxidase contains three coppers. Two copper atoms bound to subunit 2 constitute the CuA site, the primary acceptor of the electrons from ferrocytochrome c . The third copper is associated with the high-spin heme A group of subunit 1. COX-de fi cient mutants of yeast have provided new clues about copper homeostasis in mitochondria. Three genes have been implicated in mitochondrial copper metabolism. COX17 codes for a low molecular copper protein present in the cytosol and the mitochondrial intermembrane space. Mutations in this gene induce a COX de fi ciency that is partially rescued by inclusion of elevated concentrations of copper in the growth medium (Glerum et al., 1996a). Since the defect in cox17 appears to be con fi ned to COX, Cox17p targets copper speci fi cally to mitochondria (Glerum et al., 1996a). The second protein, Sco1p, is an inner membrane protein facing the intermembrane space. Mutations in SCO1 lead to a speci fi c COX de fi ciency (Schulze and Rodel, 1988). Subsequently, SCO1 was shown to be a high copy suppressor of cox17 (Glerum et al., 1996b). The genetic interaction of SCO1 and COX17 suggested that the two proteins function in a common pathway. Sco1p has a domain with sequence similarity to the copper-binding site of subunit 2. The functional importance of this region was demonstrated by the loss of Sco1p function when each of the cysteine residues in the presumed copper-binding CxxxC motif was changed to alanine by site-directed mutagenesis (Rentzsch et al., 1999). A role of Sco1p in subunit 2 maturation gains further support from antibody pull-down experi- ments demonstrating a complex of the two proteins (Lode et al., 2000). Sco1p was initially proposed to transfer copper from Cox17p to subunit 2 (Glerum et al., 1996a,b). More recently, however, an alternate function has been proposed for Sco1p based on its homology to disul fi de reductases (Chinenov, 2000). According to this interpretation, Sco1p is more likely to be involved in reduction of the cysteines (copper ligands) in subunit 2 as a prerequisite for copper binding. Neither Sco1p mediated copper transfer, or disul- fi de reduction has been demonstrated directly. The SCO2 gene of yeast is a highly conserved homolog of SCO1 (Smits et al., 1994). The function of this gene is not known but in high copy, it also suppresses cox17 mutations, although less ef fi ciently than SCO1 (Glerum et al., 1996b). Cytochrome oxidase in Rhodobacter sphaeroides is an ‘ a,a 3 ’ type enzyme with CuA and CuB sites. COX11 , a gene essential for expression of COX in yeast (Tzagoloff et al., 1990), was fi rst thought to function in heme A synthesis because of the low content of this heme in cox11 mutants (Tzagoloff et al., 1993). Recent studies of the Rhodobacter enzyme, however, indicate a role of Cox11p in the formation of the CuB and the Mg/Mn centers (Hiser et al., 2000). Rhodobacter COX puri fi ed from cox11 mutants lacks the CuB center and is depleted in Mg, even though the CuA and heme A centers are present (Hiser et al., 2000). It is obvious from this discussion that our knowledge of COX assembly is still very patchy. The temporal order of subunits interaction, the extent to which this is a protein- assisted process, and the timing of prosthetic group addition, are all questions that remain to be answered. Nor is it clear whether subunits are inserted at speci fi c membrane sites or whether they fi nd each other by lateral diffusion from different insertion sites. There are still a substantial number of COX-speci fi c nuclear genes about which almost nothing is known except that they intervene late in the assembly pathway. Mutants de fi ning this class of genes express all the COX components, import the nuclear products, but for unknown reasons are unable to complete assembly of the complex. Many existing gaps in the puzzle will be fi lled in once the functions of this class of genes are better understood. COX de fi ciency is the most frequent cause of respiratory chain defects in humans. Patients af fl icted with this disease present heterogeneous clinical phenotypes, including Leigh syndrome (Leigh, 1951), hepatic failure and encephalomyopathy (Table 2). Several factors probably contribute to the clinical heterogeneity. First, expression of the enzyme is affected by a large number of genes (Tables 1 and 3). Secondly, tissue-speci fi c differences may exist in the cellular abundance of COX-related gene products. In the case of mutations in the mitochondrial genes, the phenotype will be determined by the percentage of mutated genomes in the mitochondrial population and tissue-speci fi c differences in the threshold at which the biochemical lesions are mani- fested. As a consequence partial loss of function mutations are likely to be more severe in tissues or organs in which the concentration of the affected product is most limiting. Mutations in the three maternally inherited genes COXI , COXII and ...