Formation of the redox cofactor centers during Cox1 maturation in yeast cytochrome oxidase.

Cytochrome c oxidase (CcO) is the terminal oxidase in the oxidative phosphorylation chain within mitochondria. Mammalian CcO is a 13-subunit complex in which three mitochondrion-encoded subunits (Cox1 to Cox3) form the catalytic core (14). The catalytic core is surrounded by nucleus-encoded subunits, which confer stability to the holoenzyme and likely provide sites for the regulation of its activity (25). The fully assembled yeast holoenzyme is further organized into supercomplexes with the bc1 cytochrome c reductase (22). The catalytic core subunits contain heme and copper redox cofactors (41). Cox2 binds two copper ions, forming the binuclear CuA center that is reduced by cytochrome c. Electrons from the CuA center are transferred to a low-spin heme a center in Cox1 and subsequently to a heterobimetallic heme a-copper site, designated heme a3:CuB, where molecular oxygen is bound and reduced to water (3, 45). The heme a cofactor found in CcO differs from protoheme in that a hydroxyethylfarnesyl group replaces a vinyl moiety and a pyrrole methyl group is oxidized to a formyl substituent. Heme a synthesis is catalyzed by two successive enzymes, Cox10 and Cox15, that reside within the inner membrane (IM) (7, 21). Cox10 is a farnesyl transferase that converts protoheme to heme o. Cox15 subsequently catalyzes the oxidation of the C-8 heme methyl group in a reaction that involves matrix Yah1 ferredoxin and Arh1 ferredoxin reductase (8, 11). Yeast cells lacking Cox15 contain no heme a, but show low levels of heme o, suggesting that the activities of the two enzymes are not linked (9). Likewise, Cox15 mutations in patients exhibiting fatal infantile hypertrophic cardiomyopathy result in reduced heme a but elevated heme o levels (2). In yeast, CcO biogenesis commences with Cox1 synthesis on mitochondrial ribosomes tethered to the IM by IM-associated Pet309 and Mss51 that bind to the 5′ untranslated region (UTR) of the Cox1 transcript (29, 40, 46). Mss51 has a second function in translational elongation of Cox1, and this function occurs within high-mass Mss51 complexes (∼450 and ∼400 kDa) consisting of Mss51, Cox14, and newly synthesized Cox1 (6, 33, 34). Cox1 appears to progress from the Mss51-containing complex to downstream transient assembly complexes involving Shy1 (31, 34). Yeast cells contain another Cox1 maturation factor, Coa1, which also forms an ∼440-kDa Cox1 assembly intermediate (34). The observed interactions of Coa1 with Mss51 and Shy1 suggest that it participates in the early Cox1 maturation pathway. Information on whether Coa1 is an integral component of the Mss51- or Shy1-containing Cox1 complexes is lacking. The heme a3 cofactor center appears to be inserted in Cox1 associated with the Shy1 complex (26, 35). The evidence for heme a3 site formation within the Shy1 complex is 2-fold. First, CcO assembly stalled at CuB site formation in Cox1 or at the downstream maturation of Cox2 results in accumulation of a transient Cox1 pro-oxidant intermediate that correlates with the presence of a reactive five-coordinate heme a3 cofactor (26). The pro-oxidant heme a3:Cox1 intermediate is absent in cells lacking Shy1, Coa1, or Cox1 (35). Second, isolation of CcO in Rhodobacter or Paracoccus cells lacking Surf1 (a Shy1 ortholog) reveals an enzyme complex deficient in heme a3 but not heme a (12, 37). Shy1 is not likely a heme a3-insertase, since yeast shy1Δ cells and mutant SURF1 human cells retain 10 to 15% residual CcO activity (17, 36, 47). Rather, Shy1 may be a Cox1 chaperone stabilizing the heme a3 site during Cox1 maturation. In the absence of Shy1, it is likely that the heme a3:Cox1 assembly intermediate is destabilized and only a fraction of the intermediate progresses to the final stages of CcO maturation. CuB site formation in Cox1 requires the assembly factor Cox11. CcO isolated from Rhodobacter sphaeroides cox11Δ cells lacked CuB but contained both hemes and the CuA site (23). However, heme a3 showed an altered environment by electron paramagnetic resonance (EPR) spectroscopy, most likely due to the absence of the CuB site. Assembly of CcO in Rhodobacter differs from that in yeast in that the three-subunit core enzyme can form without heme or Cu cofactors (24). In contrast, yeast cells lacking Cox11 fail to assemble CcO, and the stalled assembly complexes are largely removed by proteolysis, although residual heme a3:Cox1 intermediates persist in cox11Δ yeast cells, resulting in hydrogen peroxide sensitivity (26). The heme a center in Cox1 may be formed earlier than the CuB-heme a3 center. Studies with fibroblasts from patients with mutant Cox10 or Cox15 reveal limited accumulation of the free Cox1 subunit (1, 2). In contrast, CcO-deficient patients with mutations in SURF1 revealed a Cox1 assembly intermediate with two nuclear CcO subunits, CoxIV and Va (equivalent to yeast subunits Cox5a and Cox6) (39, 43, 47). One interpretation of these results is that heme a insertion may be necessary for formation or stabilization of the S2 intermediate. In addition, studies of the assembly of the bo3 oxidase of Escherichia coli revealed that insertion of heme b (analogous to the heme a site in cytochrome oxidase) was necessary for subunit assembly (38). Two goals motivated the present work. First, we sought to elucidate the interrelationship of the various Cox1 maturation complexes involving Mss51, Coa1, and Shy1. Second, we wanted to discern the steps in which the heme a and CuB-heme a3 cofactor sites are formed during Cox1 maturation in yeast. We show here that separate Mss51-Cox1, Coa1-Cox1, and Shy1-Cox1 assembly intermediates exist and that the heme a and CuB centers are formed downstream of the Mss51-containing and Coa1-containing Cox1 intermediates.