Product-controlled steady-state kinetics between cytochrome aa3 from Rhodobacter sphaeroides and equine ferrocytochrome c analyzed by a novel spectrophotometric approach

Cytochrome c oxidase (CcO) catalyzes the reduction of molecular oxygen to water using ferrocytochrome c (cyt c^2 +) as the electron donor. In this study, the oxidation of horse cyt c^2 + by CcO from Rhodobacter sphaeroides, was monitored using stopped-flow spectrophotometry. A novel analytic procedure was applied in which the spectra were deconvoluted into the reduced and oxidized forms of cyt c by a least-squares fitting method, yielding the reaction rates at various concentrations of cyt c^2 + and cyt c^3 +. This allowed an analysis of the effects of cyt c^3 + on the steady-state kinetics between CcO and cyt c^2 +. The results show that cyt c^3 + exhibits product inhibition by two mechanisms: competition with cyt c^2 + at the catalytic site and, in addition, an interaction at a second site which further modulates the reaction of cyt c^2 + at the catalytic site. These results are generally consistent with previous reports, indicating the reliability of the new procedure. We also find that a 6 × His-tag at the C-terminus of the subunit II of CcO affects the binding of cyt c at both sites. The approach presented here should be generally useful in spectrophotometric studies of complex enzyme kinetics. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Differential effects of glutamate-286 mutations in the aa(3)-type cytochrome c oxidase from Rhodobacter sphaeroides and the cytochrome bo(3) ubiquinol oxidase from Escherichia coli

Both the aa(3)-type cytochrome c oxidase from Rhodobacter sphaeroides (RsCcO(aa3)) and the closely related bo(3)-type ubiquinol oxidase from Escherichia coli (EcQO(bo3)) possess a proton-conducting D-channel that terminates at a glutamic acid, E286, which is critical for controlling proton transfer to the active site for oxygen chemistry and to a proton loading site for proton pumping. E286 mutations in each enzyme block proton flux and, therefore, inhibit oxidase function. In the current work, resonance Raman spectroscopy was used to show that the E286A and E286C mutations in RsCcO(aa3) result in long range conformational changes that influence the protein interactions with both heme a and heme a(3). Therefore, the severe reduction of the steady-state activity of the E286 mutants in RsCcO(aa3) to ~0.05% is not simply a result of the direct blockage of the D-channel, but it is also a consequence of the conformational changes induced by the mutations to heme a and to the heme a(3)-Cu(B) active site. In contrast, the E286C mutation of EcQO(bo3) exhibits no evidence of conformational changes at the two heme sites, indicating that its reduced activity (3%) is exclusively a result of the inhibition of proton transfer from the D-channel. We propose that in RsCcO(aa3), the E286 mutations severely perturb the active site through a close interaction with F282, which lies between E286 and the heme-copper active site. The local structure around E286 in EcQO(bo3) is different, providing a rationale for the very different effects of E286 mutations in the two enzymes. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.     

G204D, a mutation that blocks the proton-conducting D-channel of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides

Cytochrome c oxidase uses the free energy of oxygen reduction to establish a transmembrane proton gradient. The proton-conducting D-channel in this enzyme is the major input pathway for protons which go to the binuclear center for water formation ("chemical protons") and likely the only input pathway for protons that get translocated across the lipid membrane ("pumped protons"). The D-channel starts at an acidic residue near the protein surface (D132, Rhodobacter sphaeroides numbering) and leads to another acidic residue near the binuclear center. Recent studies have shown that mutants that introduce an additional acidic residue in the channel (N139D) have the remarkable effect of accelerating steady-state oxidase activity but completely eliminating proton pumping. In this work, an aspartic acid was introduced at the position of glycine 204, G204D, which is also within the D-channel, and the effects were examined. In contrast to N139D, the G204D mutation results in a dramatic decrease of the steady-state oxygen reductase activity (<2% of wild type) [Aagaard, A., and Brzezinski, P. (2001) FEBS Lett. 494, 157-160]. The residual activity is not coupled to the proton pump, and furthermore, in reconstituted vesicles the mutant enzyme exhibits a reverse respiration control ratio; i.e., the mutant oxidase activity is stimulated rather than inhibited when working against a protonmotive force. Hence, the mutant behaves very much like the D132N, which blocks proton uptake through the D-channel. Single-turnover experiments show that the rate-limiting step in the reaction of O2 with the fully reduced G204D mutant is the F --> O transition, similar to the D132N mutant. The block of the D-channel in the D132N mutant can be partly bypassed by biochemically removing subunit III from the enzyme, indicating that removal of the subunit reveals an alternate entrance for protons to the channel. However, this is not observed with the G204D mutant. This suggests that the cryptic entrance to the D-channel that is revealed by the removal of subunit III is between the levels of G204 and D132.     

Cytochrome aa3 of Rhodobacter sphaeroides as a model for mitochondrial cytochrome c oxidase. Purification, kinetics, proton pumping, and spectral analysis.

Aerobically grown Rhodobacter sphaeroides synthesizes a respiratory chain similar to that of eukaryotes. We describe the purification of the aa3-type cytochrome c oxidase of Rb. sphaeroides as a highly active (Vmax > or = 1800 s-1), three-subunit enzyme from isolated, washed cytoplasmic membranes by hydroxylapatite chromatography and anion exchange fast protein liquid chromatography. The purified oxidase exhibits biphasic kinetics of oxidation of mammalian cytochrome c, similar to mitochondrial oxidases, and pumps protons efficiently (H+/e- = 0.7) following reconstitution into phospholipid vesicles. A membrane-bound cytochrome c is associated with the aa3-type oxidase in situ, but is removed during purification. The EPR spectra of the Rb. sphaeroides enzyme suggest the presence of a strong hydrogen bond to one or both of the histidine ligands of heme a. In other respects, optical, EPR, and resonance Raman analyses of the metal centers and their protein environments demonstrate a close correspondence between the bacterial enzyme and the structurally more complex bovine cytochrome c oxidase. The results establish this bacterial oxidase as an excellent model system for the mammalian enzyme and provide the basis for site-directed mutational analysis of its energy transducing function.     

Mobile cytochrome c2 and membrane-anchored cytochrome cy are both efficient electron donors to the cbb3- and aa3-type cytochrome c oxidases during respiratory growth of Rhodobacter sphaeroides

We have recently established that the facultative phototrophic bacterium Rhodobacter sphaeroides, like the closely related Rhodobacter capsulatus species, contains both the previously characterized mobile electron carrier cytochrome c2 (cyt c2) and the more recently discovered membrane-anchored cyt cy. However, R. sphaeroides cyt cy, unlike that of R. capsulatus, is unable to function as an efficient electron carrier between the photochemical reaction center and the cyt bc1 complex during photosynthetic growth. Nonetheless, R. sphaeroides cyt cy can act at least in R. capsulatus as an electron carrier between the cyt bc1 complex and the cbb3-type cyt c oxidase (cbb3-Cox) to support respiratory growth. Since R. sphaeroides harbors both a cbb3-Cox and an aa3-type cyt c oxidase (aa3-Cox), we examined whether R. sphaeroides cyt cy can act as an electron carrier to either or both of these respiratory terminal oxidases. R. sphaeroides mutants which lacked either cyt c2 or cyt cy and either the aa3-Cox or the cbb3-Cox were obtained. These double mutants contained linear respiratory electron transport pathways between the cyt bc1 complex and the cyt c oxidases. They were characterized with respect to growth phenotypes, contents of a-, b-, and c-type cytochromes, cyt c oxidase activities, and kinetics of electron transfer mediated by cyt c2 or cyt cy. The findings demonstrated that both cyt c2 and cyt cy are able to carry electrons efficiently from the cyt bc1 complex to either the cbb3-Cox or the aa3-Cox. Thus, no dedicated electron carrier for either of the cyt c oxidases is present in R. sphaeroides. However, under semiaerobic growth conditions, a larger portion of the electron flow out of the cyt bc1 complex appears to be mediated via the cyt c2-to-cbb3-Cox and cyt cy-to-cbb3-Cox subbranches. The presence of multiple electron carriers and cyt c oxidases with different properties that can operate concurrently reveals that the respiratory electron transport pathways of R. sphaeroides are more complex than those of R. capsulatus.     

Multiwavelength analysis of the kinetics of reduction of cytochrome aa3 by cytochrome c.

Some new approaches to the kinetic study of the reduction of cytochrome aa3 by cytochrome c are presented. The primary innovations are the use of a spectrometer which can acquire multiwavelength data as fast as every 10 microseconds, and the application of a variety of analytical methods which can utilize simultaneously all of the time-resolved spectral data. These techniques include singular value decomposition (SVD), deconvolutions based on pure Gaussian models for absorption peaks, deconvolutions based on isolated absorption spectra for the pure components, and simulations of SVD-deduced and actual experimental difference spectra. The reduction characteristics of the anaerobic resting enzyme can be distinguished from those of pulsed forms. In the former case, only two electrons can be bound by cytochrome aa3, whereas in the latter case complete reduction of the enzyme is achieved.     

Definition of the interaction domain for cytochrome c on cytochrome c oxidase. I. Biochemical, spectral, and kinetic characterization of surface mutants in subunit ii of Rhodobacter sphaeroides cytochrome aa(3)

To determine the interaction site for cytochrome c (Cc) on cytochrome c oxidase (CcO), a number of conserved carboxyl residues in subunit II of Rhodobacter sphaeroides CcO were mutated to neutral forms. A highly conserved tryptophan, Trp(143), was also mutated to phenylalanine and alanine. Spectroscopic and metal analyses of the surface carboxyl mutants revealed no overall structural changes. The double mutants D188Q/E189N and D151Q/E152N exhibit similar steady-state kinetic behavior as wild-type oxidase with horse Cc and R. sphaeroides Cc(2), showing that these residues are not involved in Cc binding. The single mutants E148Q, E157Q, D195N, and D214N have decreased activities and increased K(m) values, indicating they contribute to the Cc:CcO interface. However, their reactions with horse and R. sphaeroides Cc are different, as expected from the different distribution of surface lysines on these cytochromes c. Mutations at Trp(143) severely inhibit activity without changing the K(m) for Cc or disturbing the adjacent Cu(A) center. From these data, we identify a Cc binding area on CcO with Trp(143) and Asp(214) close to the site of electron transfer and Glu(148), Glu(157), and Asp(195) providing electrostatic guidance. The results are completely consistent with time-resolved kinetic measurements (Wang, K., Zhen, Y., Sadoski, R., Grinnell, S., Geren, L., Ferguson-Miller, S., Durham, B., and Millett, F. (1999) J. Biol. Chem. 274, 38042-38050) and computational docking analysis (Roberts, V. A., and Pique, M. E. (1999) J. Biol. Chem. 274, 38051-38060).     

Characterization of Rhodobacter sphaeroides cytochrome c(2) proteins with altered heme attachment sites

In c-type cytochromes, heme is attached to the polypeptide via thioether linkages between vinyl groups on the tetrapyrrole ring and cysteine thiols in a CX(2)CH motif. To study the role of the heme-binding site in c-type cytochrome assembly and function, we generated amino acid changes in this region of Rhodobacter sphaeroides cytochrome c(2) ((15)Cys-Gln-Thr-Cys-His(19)). Amino acid substitutions at Cys(15), Cys(18), or His(19) produced mutant proteins that did not support growth via photosynthesis where this electron carrier is required. Many of these changes appeared to slow signal peptide removal, suggesting that heme attachment is coupled to processing of the c-type cytochrome precursor protein. Inserting an alanine between the cysteine ligands (CycA-Ins17A) did not significantly alter the behavior of this protein in vivo and in vitro, suggesting that the existence of 2 residues between cysteine thiols is not essential for heme attachment to a Class I c-type cytochrome like cytochrome c(2).     

Interactions between cytochrome c2 and the photosynthetic reaction center from Rhodobacter sphaeroides: the cation-pi interaction

The cation-pi interaction between positively charged and aromatic groups is a common feature of many proteins and protein complexes. The structure of the complex between cytochrome c(2) (cyt c(2)) and the photosynthetic reaction center (RC) from Rhodobacter sphaeroides exhibits a cation-pi complex formed between Arg-C32 on cyt c(2) and Tyr-M295 on the RC [Axelrod, H. L., et al. (2002) J. Mol. Biol. 319, 501-515]. The importance of the cation-pi interaction for binding and electron transfer was studied by mutating Tyr-M295 and Arg-C32. The first- and second-order rates for electron transfer were not affected by mutating Tyr-M295 to Ala, indicating that the cation-pi complex does not greatly affect the association process or structure of the state active in electron transfer. The dissociation constant K(D) showed a greater increase when Try-M295 was replaced with nonaromatic Ala (3-fold) as opposed to aromatic Phe (1.2-fold), which is characteristic of a cation-pi interaction. Replacement of Arg-C32 with Ala increased K(D) (80-fold) largely due to removal of electrostatic interactions with negatively charged residues on the RC. Replacement with Lys increased K(D) (6-fold), indicating that Lys does not form a cation-pi complex. This specificity for Arg may be due to a solvation effect. Double mutant analysis indicates an interaction energy between Tyr-M295 and Arg-C32 of approximately -24 meV (-0.6 kcal/mol). This energy is surprisingly small considering the widespread occurrence of cation-pi complexes and may be due to the tradeoff between the favorable cation-pi binding energy and the unfavorable desolvation energy needed to bury Arg-C32 in the short-range contact region between the two proteins.     

The X-ray crystal structures of wild-type and EQ(I-286) mutant cytochrome c oxidases from Rhodobacter sphaeroides

Historically referred to as "the GTPase center", the L11 binding region (L11BR) of Escherichia coli 23 S rRNA is a highly conserved structure that has been implicated in several essential functions during protein synthesis. Here, in vivo expression of an RNA fragment containing that structure was found to affect translation termination in a codon-specific manner. The cause of these effects appeared to be titration of ribosomal protein L11, since normal phenotypes could be restored by simultaneous overproduction of wild-type L11 but not mutant L11. Subsequently, altered termination phenotypes were produced when the availability of L11 was limited by overexpression of RNA antisense to L11 mRNA and, finally, by inactivation of the chromosomal L11 gene, and they too were reversible by simultaneous expression of cloned L11. Our results indicate that in the intact cell the L11BR is an integral functional unit important for translation termination and that the presence of L11 in ribosomes is required for UAG-dependent termination and is somewhat inhibitory of UGA-dependent termination.     

Regulation of a cytochrome c2 isoform in wild-type and cytochrome c2 mutant strains of Rhodobacter sphaeroides.

In Rhodobacter sphaeroides, mutations that suppress the photosynthetic deficiency (spd mutations) of strains lacking cytochrome c2 (cyt c2) cause accumulation of a periplasmic cyt c2 isoform that has been designated isocytochrome c2 (isocyt c2). In this study, a new method for purification of both cyt c2 and isocyt c2 is described that uses periplasmic fluid as a starting material. In addition, antiserum to isocyt c2 has been used to demonstrate that all suppressor mutants contain an isocyt c2 of approximately 15 kDa. Western blot analysis indicates that isocyt c2 was present at lower levels in both wild-type and cyt c2 mutants than in spd-containing mutants. Although isocyt c2 is detectable under all growth conditions in wild-type cells, the highest level of isocyt c2 is present under aerobic conditions. Our results demonstrate that spd mutations increase the steady state level of isocyt c2 under photosynthetic conditions. Although the physiological function of isocyt c2 in wild-type cells is not known, we show that a nitrate-regulated protein in Rhodobacter sphaeroides f. sp. denitrificans also reacts with the isocyt c2 antiserum.     

Generation,characterization and crystallization of a cytochrome c1-subunit IV fused cytochrome bc1 complex from Rhodobacter sphaeroides

Cytochrome bc₁ complex catalyzes the reaction of electron transfer from ubiquinol to cytochrome c (or cytochrome c₂) and couples this reaction to proton translocation across the membrane. Crystallization of the Rhodobacter sphaeroides bc₁ complex resulted in crystals containing only three core subunits. To mitigate the problem of subunit IV being dissociated from the three-subunit core complex during crystallization, we recently engineered an R. sphaeroides mutant in which the N-terminus of subunit IV was fused to the C-terminus of cytochrome c₁ with a 14-glycine linker between the two fusing subunits, and a 6-histidine tag at the C-terminus of subunit IV (c₁-14Gly-IV-6His). The purified fusion mutant complex shows higher electron transfer activity, more structural stability, and less superoxide generation as compared to the wild-type enzyme. Preliminary crystallization attempts with this mutant complex yielded crystals containing four subunits and diffracting X-rays to 5.5 Å resolution.     

Pathways of cytochrome c oxidation by soluble and membrane-bound cytochrome aa3

In media of low ionic strength, membraneous cytochrome c oxidase, isolated cytochrome c oxidase, and proteoliposomal cytochrome c oxidase each bind cytochrome c at two sites, one of low affinity (1 microM greater than Kd' greater than 0.2 microM) and readily reversible and the other of high affinity (0.01 microM greater than Kd) and weakly reversible. When cytochrome c occupies both sites, including the low affinity site, the maximal turnover measured polarographically with ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) is independent of TMPD concentration, and lies between 250 and 400 s-1 (30 degrees C, pH 7.4) for fully activated systems. The apparent affinity of the enzyme for cytochrome c is, however, TMPD dependent. When cytochrome c occupies only the high-affinity site, the maximal turnover is closely dependent upon the concentration of TMPD, which, unlike ascorbate, can reduce bound cytochrome c. As TMPD concentration is increased, the maximal turnover approaches that seen when both sites as occupied. The lower activity of isolated cytochrome aa3 is due to the presence of inactive or inaccessible enzyme molecules. Incorporation of isolated enzyme into phospholipid vesicles restores full activity to all the subsequently accessible cytochrome aa3 molecules. Negatively charged (asolectin) vesicles show a higher cytochrome c affinity at the low-affinity sites than do the other enzyme preparations. A model for the cytochrome c-cytochrome aa3 complexes is put forward in which both sites, when occupied, are fully catalytically competent, but in which occupation of the "tight" site by a catalytically functional cytochrome c molecule is required for overall oxidation of cytochrome c via the "loose" site.     

Replacing Asn207 by aspartate at the neck of the D channel in the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides results in decoupling the proton pump

Cytochrome oxidase catalyzes the reduction of O2 to water and conserves the considerable free energy available from this reaction in the form of a proton motive force. For each electron, one proton is electrogenically pumped across the membrane. Of particular interest is the mechanism by which the proton pump operates. Previous studies of the oxidase from Rhodobacter sphaeroides have shown that all of the pumped protons enter the enzyme through the D channel and that a point mutant, N139D, in the D channel completely eliminates proton pumping without reducing oxidase activity. N139 is one of three asparagines near the entrance of the D channel, where there is a narrowing or neck, through which a single file of water molecules pass. In the current work, it is shown that replacement of a second asparagine in this region by an asparate, N207D, also decouples the proton pump without altering the oxidase activity of the enzyme. Previous studies demonstrated that the N139D mutant results in an increase in the apparent pKa of E286, a functionally critical residue that is located 20 A away from N139 at the opposite end of the D channel. In the current work, it is shown that the N207 mutation also increases the apparent pKa of E286. This finding reinforces the proposal that the elimination of proton pumping is the result of an increase of the apparent proton affinity of E286, which, in turn, prevents the timely proton transfer to a proton accepter group within the exit channel of the proton pump.     

The rate-limiting step and nonhyperbolic kinetics in the oxidation of ferrocytochrome c catalyzed by cytochrome c oxidase

The level of reduction of cytochrome a and CuA during the oxidation of ferrocytochrome c has been determined in stopped-flow experiments. Both components are partially reduced but become progressively more oxidized as the reaction proceeds. When all cytochrome c has been oxidized, CuA is also completely oxidized, whereas cytochrome a is still partially reduced. These results can be simulated on the basis of a model which requires that the intramolecular electron transfer from cytochrome a and CuA to cytochrome a3-CuB is a two-electron process and, in addition, that the binding of oxidized cytochrome c to the electron- transfer site decreases the rate constants for intramolecular electron transfer from cytochrome a. The first requirement is related to the function of the oxidase as a proton pump. Product dissociation is not by itself rate-limiting, making it less likely that the source of the nonhyperbolic substrate kinetics is an effect on this step from electrostatic interaction with ferricytochrome c bound to a second site. It is pointed out that nonhyperbolic kinetics is, in fact, an intrinsic property of ion pumps.     

The reaction of cytochrome aa3 with (porphyrin) cytochrome c as studied by pulse radiolysis

(1) Using the pulse-radiolysis and stopped-flow techniques, the reactions of iron-free (porphyrin) cytochrome c and native cytochrome c with cytochrome aa3 were investigated. The porphyrin cytochrome c anion radical (generated by reduction of porphyrin cytochrome c by the hydrated electron) can transfer its electron to cytochrome aa3. The bimolecular rate constant for this reaction is 2 x 10(7) M-1 . s-1 (5 mM potassium phosphate, 0.5% Tween 20, pH 7.0, 20 degrees C). (2) The ionic strength dependence of the cytochrome c-cytochrome aa3 interaction was measured in the ionic strength range between 40 and 120 mM. At ionic strengths below 30 mM, a cytochrome c-cytochrome aa3 complex is formed in which cytochrome c is no longer reducible by the hydrated electron. A method is described by which the contributions of electrostatic forces to the reaction rate can be determined. (3) Using the stopped-flow technique, the effect of the dielectric constant (epsilon) of the reaction medium on the reaction of cytochrome C with cytochrome aa3 was investigated. With increasing epsilon the second-order rate constant decreased.     

Blocking the K-pathway still allows rapid one-electron reduction of the binuclear center during the anaerobic reduction of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides

The K-pathway is one of the two proton-input channels required for function of cytochrome c oxidase. In the Rhodobacter sphaeroides cytochrome c oxidase, the K-channel starts at Glu101 in subunit II, which is at the surface of the protein exposed to the cytoplasm, and runs to Tyr288 at the heme a₃/Cu_B active site. Mutations of conserved, polar residues within the K-channel block or inhibit steady state oxidase activity. A large body of research has demonstrated that the K-channel is required to fully reduce the heme/Cu binuclear center, prior to the reaction with O₂, presumably by providing protons to stabilize the reduced metals (ferrous heme a₃ and cuprous Cu_B). However, there are conflicting reports which raise questions about whether blocking the K-channel blocks both electrons or only one electron from reaching the heme/Cu center. In the current work, the rate and extent of the anaerobic reduction of the heme/Cu center were monitored by optical and EPR spectroscopies, comparing the wild type and mutants that block the K-channel. The new data show that when the K-channel is blocked, one electron will still readily enter the binuclear center. The one-electron reduction of the resting oxidized (“O”) heme/Cu center of the K362M mutant, results in a partially reduced binuclear center in which the electron is distributed about evenly between heme a₃ and Cu_B in the R. sphaeroides oxidase. Complete reduction of the heme/Cu center requires the uptake of two protons which must be delivered through the K-channel.     

The roles of Rhodobacter sphaeroides copper chaperones PCuAC and Sco (PrrC) in the assembly of the copper centers of the aa3-type and the cbb3-type cytochrome c oxidases

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 Cu_A center and mono-copper Cu_B, plus a cbb₃-type CcO that contains Cu_B but lacks Cu_A. Three copper chaperones are located in the periplasm of R. sphaeroides, PCu_AC, PrrC (Sco) and Cox11. Cox11 is required to assemble Cu_B of the aa₃-type but not the cbb₃-type CcO. PrrC is homologous to mitochondrial Sco1; Sco proteins are implicated in Cu_A assembly in mitochondria and bacteria, and with Cu_B assembly of the cbb₃-type CcO. PCu_AC is present in many bacteria, but not mitochondria. PCu_AC of Thermus thermophilus metallates a Cu_A 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 PCu_AC, PrrC, or both. The absence of either chaperone strongly lowers the accumulation of both CcOs in the cells grown in low concentrations of Cu^2 +. The absence of PrrC has a greater effect than the absence of PCu_AC and PCu_AC appears to function upstream of PrrC. Analysis of purified aa₃-type CcO shows that PrrC has a greater effect on the assembly of its Cu_A than does PCu_AC, and both chaperones have a lesser but significant effect on the assembly of its Cu_B even though Cox11 is present. Scenarios for the cellular roles of PCu_AC and PrrC are considered. The results are most consistent with a role for PrrC in the capture and delivery of copper to Cu_A of the aa₃-type CcO and to Cu_B of the cbb₃-type CcO, while the predominant role of PCu_AC may be to capture and deliver copper to PrrC and Cox11. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.