Differential affinity of BsSCO for Cu(II) and Cu(I) suggests a redox role in copper transfer to the Cu(A) center of cytochrome c oxidase

SCO (synthesis of cytochrome c oxidase) proteins are involved in the assembly of the respiratory chain enzyme cytochrome c oxidase acting to assist in the assembly of the Cu(A) center contained within subunit II of the oxidase complex. The Cu(A) center receives electrons from the reductive substrate ferrocytochrome c, and passes them on to the cytochrome a center. Cytochrome a feeds electrons to the oxygen reaction site composed of cytochrome a(3) and Cu(B). Cu(A) consists of two copper ions positioned within bonding distance and ligated by two histidine side chains, one methionine, a backbone carbonyl and two bridging cysteine residues. The complex structure and redox capacity of Cu(A) present a potential assembly challenge. SCO proteins are members of the thioredoxin family which led to the early suggestion of a disulfide exchange function for SCO in Cu(A) assembly, whereas the copper binding capacity of the Bacillus subtilis version of SCO (i.e., BsSCO) suggests a direct role for SCO proteins in copper transfer. We have characterized redox and copper exchange properties of apo- and metalated-BsSCO. The release of copper (II) from its complex with BsSCO is best achieved by reducing it to Cu(I). We propose a mechanism involving both disulfide and copper exchange between BsSCO and the apo-Cu(A) site. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

Copper binding traps the folded state of the SCO protein from Bacillus subtilis

The SCO protein from the aerobic bacterium Bacillus subtilis (BsSCO) is involved in the assembly of the cytochrome c oxidase complex, and specifically with the Cu_A center. BsSCO has been proposed to play various roles in Cu_A assembly including, the direct delivery of copper ions to the Cu_A site, and/or maintaining the appropriate redox state of the cysteine ligands during formation of Cu_A. BsSCO binds copper in both Cu(II) and Cu(I) redox states, but has a million-fold higher affinity for Cu(II). As a prerequisite to kinetic studies, we measured equilibrium stability of oxidized, reduced and Cu(II)-bound BsSCO by chemical and thermal induced denaturation. Oxidized and reduced apo-BsSCO exhibit two-state behavior in both chemical- and thermal-induced unfolding. However, the Cu(II) complex of BsSCO is stable in up to nine molar urea. Thermal or guanidinium-induced unfolding of BsSCO-Cu(II) ensues only as the Cu(II) species is lost. The effect of copper (II) on the folding of BsSCO is complicated by a rapid redox reaction between copper and reduced, denatured BsSCO. When denatured apo-BsSCO is refolded in the presence of copper (II) some of the population is recovered as the BsSCO-Cu(II) complex and some is oxidized indicating that refolding and oxidation are competing processes. The proposed functional roles for BsSCO in vivo require that its cysteine residues are reduced and the presence of copper during folding may be detrimental to BsSCO attaining its functional state.     

Role of cooperative H(+)/e(-) linkage (redox bohr effect) at heme a/Cu(A) and heme a(3)/Cu(B) in the proton pump of cytochrome c oxidase

It is a pleasure to contribute to the special issue published in honor of Vladimir Skulachev, a distinguished scientist who greatly contributes to maintain a high standard of biochemical research in Russia. A more particular reason can be found in his work (Artzabanov, V. Y., Konstantinov, A. A., and Skulachev, V. P. (1978) FEBS Lett., 87, 180–185), where observations anticipating some ideas presented in my article were reported. Cytochrome c oxidase exhibits protonmotive, redox linked allosteric cooperativity. Experimental observations on soluble bovine cytochrome c oxidase are presented showing that oxido-reduction of heme a/Cu_A and heme a ₃/Cu_B is linked to deprotonation/protonation of two clusters of protolytic groups, A₁ and A₂, respectively. This cooperative linkage (redox Bohr effect) results in the translocation of 1 H⁺/oxidase molecule upon oxido-reduction of heme a/Cu_A and heme a ₃/Cu_B, respectively. Results on liposome-reconstituted oxidase show that upon oxidation of heme a/Cu_A and heme a ₃/Cu_B protons from A₁ and A₂ are released in the outer aqueous phase. A₁ but not A₂ appears to take up protons from the inner aqueous space upon reduction of the respective redox center. A cooperative model is presented in which the A₁ and A₂ clusters, operating in close sequence, constitute together the gate of the proton pump in cytochrome c oxidase.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 220–230.Original Russian Text Copyright © 2005 by Papa.This revised version was published online in April 2005 with corrections to the post codes.     

Reactivity of ligand-swapped mutants of the SCO protein from Bacillus subtilis. Isomers of the CCH metal binding motif

The Synthesis of Cytochrome Oxidase protein, or SCO protein, is required for the assembly of cytochrome c oxidase in many mitochondrial and bacterial respiratory chains. SCOs have been proposed to deliver copper to the Cu_A site of cytochrome c oxidase. We have reported that Bacillus subtilis SCO (i.e., BsSCO) binds Cu(II) with high-affinity via a two-step process mediated by three conserved residues (i.e., two cysteines and one histidine, or the CCH motif). A remarkable feature in the reaction of reduced (i.e., di-thiol) BsSCO with copper is that it does not generate any of the disulfide form of BsSCO. This molecular aversion is proposed to be a consequence of a binding mechanism in which the initial copper complex of BsSCO does not involve cysteine, but instead involves nitrogen ligands. We test this proposal here by constructing two isomers of BsSCO in which the conserved copper binding residues (i.e., the CCH-motif) are retained, but their positions are altered. In these variants the two cysteines are exchanged with histidine, and both react transiently with copper (II) with distinct kinetic profiles. The reaction generates Cu(I) and the protein is oxidized to its disulfide form. EPR analysis supports a copper binding model in which cysteine, which is at the “histidine position” in the mutant, is part of an initial encounter complex with copper. When cysteine is the initial ligating residue an oxidation reaction ensues. In contrast initial binding to native BsSCO uses nitrogen-based ligands, and thereby avoids the opportunity for thiol oxidation.     

Avoiding premature oxidation during the binding of Cu(II) to a dithiolate site in BsSCO. A rapid freeze-quench EPR study

The Bacillus subtilis version of SCO1 (BsSCO) is required for assembly of Cu(A) in cytochrome c oxidase and may function in thiol-disulfide exchange and/or copper delivery. BsSCO binds Cu(II) with ligation by two cysteines, one histidine and one water. However, copper is a catalyst of cysteine oxidation and BsSCO must avoid this reaction to remain functional. Time resolved, rapid freeze-quench (RFQ) electron paramagnetic resonance of apo-BsSCO reacting with Cu(II) reveals an initial Cu(II) species with two equatorially coordinated nitrogen atoms, but no sulfur. We propose that BsSCO evolves from this initial sulfur free coordination of Cu(II) to the final dithiolate species via a change in conformation, and that the initial binding by nitrogen is a means for BsSCO to avoid premature thiol oxidation.     

Copper binding traps the folded state of the SCO protein from Bacillus subtilis

The SCO protein from the aerobic bacterium Bacillus subtilis (BsSCO) is involved in the assembly of the cytochrome c oxidase complex, and specifically with the Cu(A) center. BsSCO has been proposed to play various roles in Cu(A) assembly including, the direct delivery of copper ions to the Cu(A) site, and/or maintaining the appropriate redox state of the cysteine ligands during formation of Cu(A). BsSCO binds copper in both Cu(II) and Cu(I) redox states, but has a million-fold higher affinity for Cu(II). As a prerequisite to kinetic studies, we measured equilibrium stability of oxidized, reduced and Cu(II)-bound BsSCO by chemical and thermal induced denaturation. Oxidized and reduced apo-BsSCO exhibit two-state behavior in both chemical- and thermal-induced unfolding. However, the Cu(II) complex of BsSCO is stable in up to nine molar urea. Thermal or guanidinium-induced unfolding of BsSCO-Cu(II) ensues only as the Cu(II) species is lost. The effect of copper (II) on the folding of BsSCO is complicated by a rapid redox reaction between copper and reduced, denatured BsSCO. When denatured apo-BsSCO is refolded in the presence of copper (II) some of the population is recovered as the BsSCO-Cu(II) complex and some is oxidized indicating that refolding and oxidation are competing processes. The proposed functional roles for BsSCO in vivo require that its cysteine residues are reduced and the presence of copper during folding may be detrimental to BsSCO attaining its functional state.     

Electron transfer after flash photolysis of mixed-valence carboxycytochrome c oxidase

The light-induced difference spectra of the fully reduced (a³⁺a²⁺₃-CO) complex and the mixed-valence carboxycytochrome c oxidase (a³⁺a²⁺₃-CO) during steady-state illumination and after flash photolysis showed marked differences. The differences appear to be due to electron transfer between the redox centres in the enzyme. The product of the absorbance coefficient and the quantum yield was found to be equal in both enzyme species, both when determined from the rates of photolysis and from the values of the dissociation constants of the cytochrome a²⁺₃-CO complex. This would confirm that the spectral properties of cytochrome a₃ are not affected by the redox state of cytochrome a and Cu_A. When the absorbance changes after photolysis of cytochrome a²⁺₃-CO with a laser flash were followed on a time scale from 1 μs to 1 s in the fully reduced carboxycytochrome c oxidase, only the CO recombination reaction was observed. However, in the mixed-valence enzyme an additional fast absorbance change (k = 7·10³s^?1) was detected. The kinetic difference spectrum of this fast change showed a peak at 415 nm and a trough at 445 nm, corresponding to oxidation of cytochrome a₃. Concomitantly, a decrease of the 830 nm band was observed due to reduction of Cu_A. This demonstrates that in the partially reduced enzyme a pathway is present between Cu_A and the cytochrome a₃-Cu_B pair, via which electrons are transferred rapidly.     

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.     

How Periplasmic Thioredoxin TlpA Reduces Bacterial Copper Chaperone ScoI and Cytochrome Oxidase Subunit II (CoxB) Prior to Metallation

Two critical cysteine residues in the copper-A site (Cu_A) on subunit II (CoxB) of bacterial cytochrome c oxidase lie on the periplasmic side of the cytoplasmic membrane. As the periplasm is an oxidizing environment as compared with the reducing cytoplasm, the prediction was that a disulfide bond formed between these cysteines must be eliminated by reduction prior to copper insertion. We show here that a periplasmic thioredoxin (TlpA) acts as a specific reductant not only for the Cu²⁺ transfer chaperone ScoI but also for CoxB. The dual role of TlpA was documented best with high-resolution crystal structures of the kinetically trapped TlpA-ScoI and TlpA-CoxB mixed disulfide intermediates. They uncovered surprisingly disparate contact sites on TlpA for each of the two protein substrates. The equilibrium of CoxB reduction by TlpA revealed a thermodynamically favorable reaction, with a less negative redox potential of CoxB (E′₀ = −231 mV) as compared with that of TlpA (E′₀ = −256 mV). The reduction of CoxB by TlpA via disulfide exchange proved to be very fast, with a rate constant of 8.4 × 10⁴ m⁻¹ s⁻¹ that is similar to that found previously for ScoI reduction. Hence, TlpA is a physiologically relevant reductase for both ScoI and CoxB. Although the requirement of ScoI for assembly of the Cu_A-CoxB complex may be bypassed in vivo by high environmental Cu²⁺ concentrations, TlpA is essential in this process because only reduced CoxB can bind copper ions.     

The sequence of electron carriers in the reaction of cytochromec oxidase with oxygen

Kinetic studies of the electron transfer processes performed by cytochrome oxidase have assigned rates of electron transfer between the metal centers involved in the oxidation of ferrocytochromec by molecular oxygen. Transient-state studies of the reaction with oxygen have led to the proposal of a sequence of carriers from cytochromec, to Cu_A, to cytochromea, and then to the binuclear (i.e., cytochromea ₃-Cu_B) center. Electron exchange rates between these centers agree with relative center-to-center distances as follows; cytochromec to Cu_A 5–7 Å, cytochromec to cytochromea 20–25 Å, Cu_A to cytochromea 14–16 Å and cytochromea to cytochrome a₃-Cu_B 8–10 Å. It is proposed that the step from cytochromea to the binuclear center is the key control point in the reaction and that this step is one of the major points of energy transduction in the reaction cycle.     

The kinetics of electron entry in cytochromec oxidase

Summary The kinetics of electron entry in beef heart cytochromec oxidase have been studied by stopped-flow spectroscopy following chemical modification of the Cu_A site with mercurials. In this derivative Cu_A is no longer reducible by cytochrome c while cytochromea may accept electrons from the latter with rates comparable to the native enzyme. The results indicate that Cu_A is not the exclusive electron entry site in cytochromec oxidase.     

Sco proteins are involved in electron transfer processes

Sco proteins are widespread in eukaryotic and in many prokaryotic organisms. They have a thioredoxin-like fold and bind a single copper(I) or copper(II) ion through a CXXXC motif and a conserved His ligand, with both tight and weak affinities. They have been implicated in the assembly of the Cu_A site of cytochrome c oxidase as copper chaperones and/or thioredoxins. In this work we have structurally characterized a Sco domain which is naturally fused with a typical electron transfer molecule, i.e., cytochrome c, in Pseudomonas putida. The thioredoxin-like Sco domain does not bind copper(II), binds copper(I) with weak affinity without involving the conserved His, and has redox properties consisting of a thioredoxin activity and of the ability of reducing copper(II) to copper(I), and iron(III) to iron(II) of the cytochrome c domain. These findings indicate that the His ligand coordination is the discriminating factor for introducing a metallochaperone function in a thioredoxin-like fold, typically responsible for electron transfer processes. A comparative structural analysis of the Sco domain from P. putida versus eukaryotic Sco proteins revealed structural determinants affecting the formation of a tight-affinity versus a weak-affinity copper binding site in Sco proteins.     

Concerted involvement of cooperative proton–electron linkage and water production in the proton pump of cytochrome c oxidase

In cytochrome c oxidase, oxido-reductions of heme a/Cu_A and heme a₃/Cu_B are cooperatively linked to proton transfer at acid/base groups in the enzyme. H⁺/e⁻ cooperative linkage at Fe_a3/Cu_B is envisaged to be involved in proton pump mechanisms confined to the binuclear center. Models have also been proposed which involve a role in proton pumping of cooperative H⁺/e⁻ linkage at heme a (and Cu_A). Observations will be presented on: (i) proton consumption in the reduction of molecular oxygen to H₂O in soluble bovine heart cytochrome c oxidase; (ii) proton release/uptake associated with anaerobic oxidation/reduction of heme a/Cu_A and heme a₃/Cu_B in the soluble oxidase; (iii) H⁺ release in the external phase (i.e. H⁺ pumping) associated with the oxidative (R → O transition), reductive (O → R transition) and a full catalytic cycle (R → O → R transition) of membrane-reconstituted cytochrome c oxidase. A model is presented in which cooperative H⁺/e⁻ linkage at heme a/Cu_A and heme a₃/Cu_B with acid/base clusters, C₁ and C₂ respectively, and protonmotive steps of the reduction of O₂ to water are involved in proton pumping.     

Re-evaluation of the near infrared spectra of mitochondrial cytochrome c oxidase: Implications for non invasive in vivo monitoring of tissues

We re-determined the near infrared (NIR) spectral signatures (650–980 nm) of the different cytochrome c oxidase redox centres, in the process separating them into their component species. We confirm that the primary contributor to the oxidase NIR spectrum between 700 and 980 nm is cupric Cu_A, which in the beef heart enzyme has a maximum at 835 nm. The 655 nm band characterises the fully oxidised haem a₃/Cu_B binuclear centre; it is bleached either when one or more electrons are added to the binuclear centre or when the latter is modified by ligands. The resulting ‘perturbed’ binuclear centre is also characterised by a previously unreported broad 715–920 nm band. The NIR spectra of certain stable liganded species (formate and CO), and the unstable oxygen reaction compounds P and F, are similar, suggesting that the latter may resemble the stable species electronically. Oxidoreduction of haem a makes no contribution either to the 835 nm maximum or the 715 nm band. Our results confirm the ability of NIRS to monitor the Cu_A centre of cytochrome oxidase activity in vivo, although noting some difficulties in precise quantitative interpretations in the presence of perturbations of the haem a₃/Cu_B binuclear centre.     

The mitochondrial TMEM177 associates with COX20 during COX2 biogenesis

The three mitochondrial-encoded proteins, COX1, COX2, and COX3, form the core of the cytochrome c oxidase. Upon synthesis, COX2 engages with COX20 in the inner mitochondrial membrane, a scaffold protein that recruits metallochaperones for copper delivery to the Cu_A-Site of COX2. Here we identified the human protein, TMEM177 as a constituent of the COX20 interaction network. Loss or increase in the amount of TMEM177 affects COX20 abundance leading to reduced or increased COX20 levels respectively. TMEM177 associates with newly synthesized COX2 and SCO2 in a COX20-dependent manner. Our data shows that by unbalancing the amount of TMEM177, newly synthesized COX2 accumulates in a COX20-associated state. We conclude that TMEM177 promotes assembly of COX2 at the level of Cu_A-site formation.     

Formamide probes a role for water in the catalytic cycle of cytochrome c oxidase

Formamide is a slow-onset inhibitor of mitochondrial cytochrome c oxidase that is proposed to act by blocking water movement through the protein. In the presence of formamide the redox level of mitochondrial cytochrome c oxidase evolves over the steady state as the apparent electron transfer rate from cytochrome a to cytochrome a₃ slows. At maximal inhibition cytochrome a and cytochrome c are fully reduced, whereas cytochrome a₃ and Cu_B remain fully oxidized consistent with the idea that formamide interferes with electron transfer between cytochrome a and the oxygen reaction site. However, transient kinetic studies show that intrinsic rates of electron transfer are unchanged in the formamide-inhibited enzyme. Formamide inhibition is demonstrated for another member of the heme-oxidase family, cytochrome c oxidase from Bacillus subtilis, but the onset of inhibition is much quicker than for mitochondrial oxidase. If formamide inhibition arises from a steric blockade of water exchange during catalysis then water exchange in the smaller bacterial oxidase is more open. Subunit III removal from the mitochondrial oxidase hastens the onset of formamide inhibition suggesting a role for subunit III in controlling water exchange during the cytochrome c oxidase reaction.     

COX19 mediates the transduction of a mitochondrial redox signal from SCO1 that regulates ATP7A-mediated cellular copper efflux

SCO1 and SCO2 are metallochaperones whose principal function is to add two copper ions to the catalytic core of cytochrome c oxidase (COX). However, affected tissues of SCO1 and SCO2 patients exhibit a combined deficiency in COX activity and total copper content, suggesting additional roles for these proteins in the regulation of cellular copper homeostasis. Here we show that both the redox state of the copper-binding cysteines of SCO1 and the abundance of SCO2 correlate with cellular copper content and that these relationships are perturbed by mutations in SCO1 or SCO2, producing a state of apparent copper overload. The copper deficiency in SCO patient fibroblasts is rescued by knockdown of ATP7A, a trans-Golgi, copper-transporting ATPase that traffics to the plasma membrane during copper overload to promote efflux. To investigate how a signal from SCO1 could be relayed to ATP7A, we examined the abundance and subcellular distribution of several soluble COX assembly factors. We found that COX19 partitions between mitochondria and the cytosol in a copper-dependent manner and that its knockdown partially rescues the copper deficiency in patient cells. These results demonstrate that COX19 is necessary for the transduction of a SCO1-dependent mitochondrial redox signal that regulates ATP7A-mediated cellular copper efflux.     

Assembly factors and ATP-dependent proteases in cytochrome c oxidase biogenesis

Eukaryotic cytochrome c oxidase (CcO), the terminal enzyme of the energy-transducing mitochondrial electron transport chain is a hetero-oligomeric, heme–copper oxidase complex composed of both mitochondrially and nuclear-encoded subunits. It is embedded in the inner mitochondrial membrane where it couples the transfer of electrons from reduced cytochrome c to molecular oxygen with vectorial proton translocation across the membrane. The biogenesis of CcO is a complicated sequential process that requires numerous specific accessory proteins, so-called assembly factors, which include translational activators, translocases, molecular chaperones, copper metallochaperones and heme a biosynthetic enzymes. Besides these CcO-specific protein factors, the correct biogenesis of CcO requires an even greater number of proteins with much broader substrate specificities. Indeed, growing evidence indicates that mitochondrial ATP-dependent proteases might play an important role in CcO biogenesis. Out of the four identified energy-dependent mitochondrial proteases, three were shown to be directly involved in proteolysis of CcO subunits. In addition to their well-established protein-quality control function these oligomeric proteolytic complexes with chaperone-like activities may function as molecular chaperones promoting productive folding and assembly of subunit proteins. In this review, we summarize the current knowledge of the functional involvement of eukaryotic CcO-specific assembly factors and highlight the possible significance for CcO biogenesis of mitochondrial ATP-dependent proteases.     

The human cytochrome c oxidase assembly factors SCO1 and SCO2 have regulatory roles in the maintenance of cellular copper homeostasis

Human SCO1 and SCO2 are metallochaperones that are essential for the assembly of the catalytic core of cytochrome c oxidase (COX). Here we show that they have additional, unexpected roles in cellular copper homeostasis. Mutations in either SCO result in a cellular copper deficiency that is both tissue and allele specific. This phenotype can be dissociated from the defects in COX assembly and is suppressed by overexpression of SCO2, but not SCO1. Overexpression of a SCO1 mutant in control cells in which wild-type SCO1 levels were reduced by shRNA recapitulates the copper-deficiency phenotype in SCO1 patient cells. The copper-deficiency phenotype reflects not a change in high-affinity copper uptake but rather a proportional increase in copper efflux. These results suggest a mitochondrial pathway for the regulation of cellular copper content that involves signaling through SCO1 and SCO2, perhaps by their thiol redox or metal-binding state.     

Effects of copper concentration in continuous culture on the aa 3-type cytochrome oxidase and respiratory chains of Paracoccus denitrificans

The role(s) of copper in a bacterial cytochrome oxidase of the aa ₃-type was investigated by growth of Paracoccus denitrificans NCIB 8944, in batch and steady state continuous culture, in a medium from which the bulk of the copper had been extracted. In a medium containing approximately 0.02 M copper, cellular copper content, cytochromes a+a ₃ and cytochrome a ₃ were reduced to 55%, 58% and 33% respectively of control values and there were also less marked decreases in cytochromes c+c ₁ (to 85%) and a CO-binding b-type cytochrome, possibly cytochrome o (to 71%). Copper deficiency elicited in reduced minus oxidized difference spectra a shift to shorter wavelengths and narrowing of the band width of the -band of the oxidase, and loss of a (negative) band near 830 nm attributable to Cu_A (the copper functionally associated with haem a in the oxidase complex). The oxidase in copper-deficient cells reacted with oxygen to form the oxy Compound A at rates similar to that in control cells but CO recombination to ferrous haem a ₃ was slowed 4-fold in the copper deficient case. The results are interpreted as indicating loss of Cu_A and changes in the proportions of haems a and a ₃ with retention of catalytic activity. Titrations of respiration rates with antimycin suggested that copper deficiency did not result in diversion of electron flux through an antimycin A-insensitive, cytochrome o-terminated branch of the respiratory chain.