Altering Conserved Lipid Binding Sites in Cytochrome c Oxidase of Rhodobacter sphaeroides Perturbs the Interaction between Subunits I and III and Promotes Suicide Inactivation of the Enzyme

Abstract

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.

Role of norEF in Denitrification, Elucidated by Physiological Experiments with Rhodobacter sphaeroides

“…However, sequence alignment of NorE with subunit III proteins reveals the conservation of a number of residues, in particular the dicyclohexylcarbodiimide (DCCD) binding glu residue found in subunit III, indicating that NorE is structurally or biochemically similar to subunit III, which has been shown to be required for oxidase stability (22,23). Even in the aa 3 oxidase family, the interaction of subunit III with subunit I is relatively weak.…”

What limits the allotopic expression of nucleus-encoded mitochondrial genes? The case of the chimeric Cox3 and Atp6 genes

“…The corresponding Chlamydomonas ATP6 and COX3 were XP_001689492 and XP_001692182. To diminish the mean hydrophobicity in TMSs, individual residues in human proteins were changed to the corresponding algal ones, but only in regions believed not to be involved in critical functions or in the interfaces with other subunits Varanasi et al, 2006). For the chimeric re-designed ATP6 (A6Chim) we added the 106 residues of the MTS followed by 13 amino acids of the mature ATP6 of Chlamydomonas.…”

“…For the chimeric human COX3 we added the MTS from Chlamydomonas COX3 plus the first 28 amino acids of the mature algal COX3. We also diminished the mean hydrophobicity in the first, fourth and sixth TMSs, without changing conserved amino acids that have been reported to be important for the assembly of Complex IV (Varanasi et al, 2006) (see Materials and methods). We also added a HA epitope in the carboxy-termini for antibody detection (COX3-HA).…”

Delipidation of cytochrome c oxidase from Rhodobacter sphaeroides destabilizes its quaternary structure

“…Studies have revealed that bound PLs affect both structural and functional integrity of membrane protein complexes, i.e. the bound PLs are essential components of the individual enzymes or supercomplexes [10,12,14–18]. …”

“…For example, CcO contains evolutionarily conserved lipid binding sites within subunit III that contain PE molecules in bacterial CcO [1], although CL can be modeled into bacterial subunit III [21], and PG in a structure of mammalian CcO [8]. Modification of these lipid binding sites within subunit III inhibits 1) the assembly of subunit III into the CcO complex, and 2) the protective function of subunit III on CcO activity even when all of the subunit is present [18]. Negatively charged PG stabilizes a dimer of photosystem II [25] similar to the role of negatively charged CL in mammalian CcO [20].…”