The catalytic core of cytochrome c oxidase is composed of three subunits where subunits I and II contain all of the redox-active metal centers and subunit III is a seven transmembrane helix protein that binds to subunit I. The N-terminal region of subunit III is adjacent to D132 of subunit I, the initial proton acceptor of the D pathway that transfers protons from the protein surface to the buried active site approximately 30 A distant. The absence of subunit III only slightly alters the initial steady-state activity of the oxidase at pH 6.5, but activity declines sharply with increasing pH, yielding an apparent pK(a) of 7.2 for steady-state O(2) reduction. When subunit III is present, cytochrome oxidase is more active at higher pH, and the apparent pK(a) of steady-state O(2) reduction is 8.5. Single-turnover experiments show that proton uptake through the D pathway at pH 8 slows from >10000 s(-1) in the presence of subunit III to 350 s(-1) in its absence. At low pH (5.5) the D pathway of the oxidase lacking subunit III regains its capacity for rapid proton uptake. Analysis of the F --> O transition indicates that the apparent pK(a) of the D pathway in the absence of subunit III is 6.8, similar to that of steady-state O(2) reduction (7.2). The pK(a) of D132 itself may decline in the absence of subunit III since its carboxylate group will be more exposed to solvent water. Alternatively, part of a proton antenna for the D pathway may be lost upon removal of subunit III. It is proposed that one role of subunit III in the normal oxidase is to maintain rapid proton uptake through the D pathway at physiologic pH.

The α proteobacter Rhodobacter sphaeroides accumulates two cytochrome c oxidases (CcO) in its cytoplasmic membrane during aerobic growth: a mitochondrial-like aa3-type CcO containing a di-copper CuA center and mono-copper CuB, plus a cbb3-type CcO that contains CuB but lacks CuA. Three copper chaperones are located in the periplasm of R. sphaeroides, PCuAC, PrrC (Sco) and Cox11. Cox11 is required to assemble CuB of the aa3-type but not the cbb3-type CcO. PrrC is homologous to mitochondrial Sco1; Sco proteins are implicated in CuA assembly in mitochondria and bacteria, and with CuB assembly of the cbb3-type CcO. PCuAC is present in many bacteria, but not mitochondria. PCuAC of Thermus thermophilus metallates a CuA center in vitro, but its in vivo function has not been explored. Here, the extent of copper center assembly in the aa3- and cbb3-type CcOs of R. sphaeroides has been examined in strains lacking PCuAC, PrrC, or both. The absence of either chaperone strongly lowers the accumulation of both CcOs in the cells grown in low concentrations of Cu2+. The absence of PrrC has a greater effect than the absence of PCuAC and PCuAC appears to function upstream of PrrC. Analysis of purified aa3-type CcO shows that PrrC has a greater effect on the assembly of its CuA than does PCuAC, and both chaperones have a lesser but significant effect on the assembly of its CuB even though Cox11 is present. Scenarios for the cellular roles of PCuAC and PrrC are considered. The results are most consistent with a role for PrrC in the capture and delivery of copper to CuA of the aa3-type CcO and to CuB of the cbb3-type CcO, while the predominant role of PCuAC may be to capture and deliver copper to PrrC and Cox11.

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 ␣-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 B active sites. 50% of the oxidase lacked heme a 3 , 10 -15% contained heme a 3 but lacked Cu B , 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.

Subunit III of the three-subunit catalytic core of cytochrome c oxidase (CcO) contains no metal centers, but it does bind two lipids, within a deep cleft, in binding sites conserved from bacteria to humans. Subunit III binds to subunit I, where it prevents the spontaneous suicide inactivation of CcO by decreasing the probability of side reactions at the heme-Cu O2 reduction site in subunit I. Subunit III prevents suicide inactivation by (1) maintaining adequate rates of proton delivery to the heme-Cu active site and (2) stabilizing the structure of the active site during turnover [Mills and Hosler (2005) Biochemistry 44, 4656]. Here, we first show that mutating several individual residues of the conserved lipid binding sites in subunit III disturbs the subunit I-III interface. Then, two lipid binding site mutants were constructed with an affinity tag on subunit III such that the mutant CcOs could be isolated with 100% subunit III. R226A eliminates an ion pair to the phosphate of the outermost lipid of the cleft, while W59A-F86A disrupts interactions with the fatty acid tails of both lipids. Once these mutant CcOs are placed into soybean phospholipid vesicles, where extensive exchange of bacterial for soybean lipids takes place, it is shown that altering the lipid binding sites mimics a major loss of subunit III, even though subunit III is completely retained, in that suicide inactivation becomes much more probable. The rate of proton delivery to the active site remains rapid, ruling out slow proton uptake as the primary reason for increased suicide inactivation upon alteration of the lipid binding sites. We conclude that altering the lipid binding sites of subunit III may promote side reactions leading to suicide inactivation by allowing greater movement to occur in and around the O2 reduction site of subunit I during the catalytic cycle.