The roles of Rhodobacter sphaeroides copper chaperones PCu(A)C and Sco (PrrC) in the assembly of the copper centers of the aa(3)-type and the cbb(3)-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 aa(3)-type CcO containing a di-copper Cu(A) center and mono-copper Cu(B), plus a cbb(3)-type CcO that contains Cu(B) but lacks Cu(A). Three copper chaperones are located in the periplasm of R. sphaeroides, PCu(A)C, PrrC (Sco) and Cox11. Cox11 is required to assemble Cu(B) of the aa(3)-type but not the cbb(3)-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 cbb(3)-type CcO. PCu(A)C is present in many bacteria, but not mitochondria. PCu(A)C 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 aa(3)- and cbb(3)-type CcOs of R. sphaeroides has been examined in strains lacking PCu(A)C, 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(A)C and PCu(A)C appears to function upstream of PrrC. Analysis of purified aa(3)-type CcO shows that PrrC has a greater effect on the assembly of its Cu(A) than does PCu(A)C, 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(A)C 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 aa(3)-type CcO and to Cu(B) of the cbb(3)-type CcO, while the predominant role of PCu(A)C 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.     

Biogenesis of cbb3-type cytochrome c oxidase in Rhodobacter capsulatus

The cbb₃-type cytochrome c oxidases (cbb₃-Cox) constitute the second most abundant cytochrome c oxidase (Cox) group after the mitochondrial-like aa₃-type Cox. They are present in bacteria only, and are considered to represent a primordial innovation in the domain of Eubacteria due to their phylogenetic distribution and their similarity to nitric oxide (NO) reductases. They are crucial for the onset of many anaerobic biological processes, such as anoxygenic photosynthesis or nitrogen fixation. In addition, they are prevalent in many pathogenic bacteria, and important for colonizing low oxygen tissues. Studies related to cbb₃-Cox provide a fascinating paradigm for the biogenesis of sophisticated oligomeric membrane proteins. Complex subunit maturation and assembly machineries, producing the c-type cytochromes and the binuclear heme b₃-Cu_B center, have to be coordinated precisely both temporally and spatially to yield a functional cbb₃-Cox enzyme. In this review we summarize our current knowledge on the structure, regulation and assembly of cbb₃-Cox, and provide a highly tentative model for cbb₃-Cox assembly and formation of its heme b₃-Cu_B binuclear center. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

Cooperation between two periplasmic copper chaperones is required for full activity of the cbb3‐type cytochrome c oxidase and copper homeostasis in Rhodobacter capsulatus

Copper (Cu) is an essential micronutrient that functions as a cofactor in several important enzymes, such as respiratory heme‐copper oxygen reductases. Yet, Cu is also toxic and therefore cells engage a highly coordinated Cu uptake and delivery system to prevent the accumulation of toxic Cu concentrations. In this study, we analyzed Cu delivery to the cbb₃‐type cytochrome c oxidase (cbb₃‐Cox) of Rhodobacter capsulatus. We identified the PCu_AC‐like periplasmic chaperone PccA and analyzed its contribution to cbb₃‐Cox assembly. Our data demonstrate that PccA is a Cu‐binding protein with a preference for Cu(I), which is required for efficient cbb₃‐Cox assembly, in particular, at low Cu concentrations. By using in vivo and in vitro cross‐linking, we show that PccA forms a complex with the Sco1‐homologue SenC. This complex is stabilized in the absence of the cbb₃‐Cox‐specific assembly factors CcoGHIS. In cells lacking SenC, the cytoplasmic Cu content is significantly increased, but the simultaneous absence of PccA prevents this Cu accumulation. These data demonstrate that the interplay between PccA and SenC not only is required for Cu delivery during cbb₃‐Cox assembly but also regulates Cu homeostasis in R. capsulatus.     

Substrate binding and the catalytic reactions in cbb3-type oxidases: The lipid membrane modulates ligand binding

Heme–copper oxidases (HCuOs) are the terminal components of the respiratory chain in the mitochondrial membrane or the cell membrane in many bacteria. These enzymes reduce oxygen to water and use the free energy from this reaction to maintain a proton-motive force across the membrane in which they are embedded. The heme–copper oxidases of the cbb₃-type are only found in bacteria, often pathogenic ones since they have a low K_m for O₂, enabling the bacteria to colonize semi-anoxic environments. Cbb₃-type (C) oxidases are highly divergent from the mitochondrial-like aa₃-type (A) oxidases, and within the heme–copper oxidase family, cbb₃ is the closest relative to the most divergent member, the bacterial nitric oxide reductase (NOR). Nitric oxide reductases reduce NO to N₂O without coupling the reaction to the generation of any electrochemical proton gradient. The significant structural differences between A- and C-type heme–copper oxidases are manifested in the lack in cbb₃ of most of the amino acids found to be important for proton pumping in the A-type, as well as in the different binding characteristics of ligands such as CO, O₂ and NO. Investigations of the reasons for these differences at a molecular level have provided insights into the mechanism of O₂ and NO reduction as well as the proton-pumping mechanism in all heme–copper oxidases. In this paper, we discuss results from these studies with the focus on the relationship between proton transfer and ligand binding and reduction. In addition, we present new data, which show that CO binding to one of the c-type hemes of CcoP is modulated by protein–lipid interactions in the membrane. These results show that the heme c-CO binding can be used as a probe of protein–membrane interactions in cbb₃ oxidases, and possible physiological consequences for this behavior are discussed.     

Stability of the cbb3-type cytochrome oxidase requires specific CcoQ-CcoP interactions

Cytochrome cbb₃-type oxidases are members of the heme copper oxidase superfamily and are composed of four subunits. CcoN contains the heme b-Cu_B binuclear center where oxygen is reduced, while CcoP and CcoO are membrane-bound c-type cytochromes thought to channel electrons from the donor cytochrome into the binuclear center. Like many other bacterial members of this superfamily, the cytochrome cbb₃-type oxidase contains a fourth, non-cofactor-containing subunit, which is termed CcoQ. In the present study, we analyzed the role of CcoQ on the stability and activity of Rhodobacter capsulatus cbb₃-type oxidase. Our data showed that CcoQ is a single-spanning membrane protein with a N_out-C_in topology. In the absence of CcoQ, cbb₃-type oxidase activity is significantly reduced, irrespective of the growth conditions. Blue native polyacrylamide gel electrophoresis analyses revealed that the lack of CcoQ specifically impaired the stable recruitment of CcoP into the cbb₃-type oxidase complex. This suggested a specific CcoQ-CcoP interaction, which was confirmed by chemical cross-linking. Collectively, our data demonstrated that in R. capsulatus CcoQ was required for optimal cbb₃-type oxidase activity because it stabilized the interaction of CcoP with the CcoNO core complex, leading subsequently to the formation of the active 230-kDa cbb₃-type oxidase complex.     

Copper Starvation-inducible Protein for Cytochrome Oxidase Biogenesis in Bradyrhizobium japonicum

Microarray analysis of Bradyrhizobium japonicum grown under copper limitation uncovered five genes named pcuABCDE, which are co-transcribed and co-regulated as an operon. The predicted gene products are periplasmic proteins (PcuA, PcuC, and PcuD), a TonB-dependent outer membrane receptor (PcuB), and a cytoplasmic membrane-integral protein (PcuE). Homologs of PcuC and PcuE had been discovered in other bacteria, namely PCu_AC and YcnJ, where they play a role in cytochrome oxidase biogenesis and copper transport, respectively. Deletion of the pcuABCDE operon led to a pleiotropic phenotype, including defects in the aa₃-type cytochrome oxidase, symbiotic nitrogen fixation, and anoxic nitrate respiration. Complementation analyses revealed that, under our assay conditions, the tested functions depended only on the pcuC gene and not on pcuA, pcuB, pcuD, or pcuE. The B. japonicum genome harbors a second pcuC-like gene (blr7088), which, however, did not functionally replace the mutated pcuC. The PcuC protein was overexpressed in Escherichia coli, purified to homogeneity, and shown to bind Cu(I) with high affinity in a 1:1 stoichiometry. The replacement of His⁷⁹, Met⁹⁰, His¹¹³, and Met¹¹⁵ by alanine perturbed copper binding. This corroborates the previously purported role of this protein as a periplasmic copper chaperone for the formation of the Cu_A center on the aa₃-type cytochrome oxidase. In addition, we provide evidence that PcuC and the copper chaperone ScoI are important for the symbiotically essential, Cu_A-free cbb₃-type cytochrome oxidase specifically in endosymbiotic bacteroids of soybean root nodules, which could explain the symbiosis-defective phenotype of the pcuC and scoI mutants.     

Two conserved non-canonical histidines are essential for activity of the cbb 3-type oxidase in Rhodobacter capsulatus

Cytochrome cbb ₃ oxidase, a member of the heme–copper oxidase superfamily, catalyses the reduction of oxygen to water and generates a proton gradient. Cytochrome c oxidases are characterized by a catalytic subunit (subunit I) containing two hemes and one copper ion ligated by six invariant histidine residues, which are diagnostic of heme–copper oxidases in all type of the heme–copper oxidase superfamily. Alignments of the amino acid sequences of subunit I (FixN or CcoN) of the cbb ₃-type oxidases show that catalytic subunit also contains six non-canonical histidine residues that are conserved in all CcoN subunits of the cbb ₃ oxidase, but not the catalytic subunits of other members of heme–copper oxidases superfamily. The function of these six CcoN-specific conserved histidines of cbb ₃-type oxidase in R. capsulatus is unknown. To analyze the contribution of the two invariant histidines of CcoN, H300 and H394, in activity and assembly of the Rhodobacter capsulatus cbb ₃-type oxidase, they were substituted for valine and alanine, respectively by site-directed mutagenesis. H300V and H394A mutations were analyzed with respect to their activity and assembly. It was found that H394A mutation led to a defect in the assembly of both CcoP and CcoO in the membrane, which results in almost complete loss of activity and that although the H300V mutant is normally assembled in the membrane and retain their stability, its catalytic activity is significantly reduced when compared with wild-type oxidase.     

Differential affinity of BsSCO for Cu(II) and Cu(I) suggests a redox role in copper transfer to the CuA 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 a₃ 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.     

Enzymatic Characterization and In Vivo Function of Five Terminal Oxidases in Pseudomonas aeruginosa

The ubiquitous opportunistic pathogen Pseudomonas aeruginosa has five aerobic terminal oxidases: bo₃-type quinol oxidase (Cyo), cyanide-insensitive oxidase (CIO), aa₃-type cytochrome c oxidase (aa₃), and two cbb₃-type cytochrome c oxidases (cbb₃-1 and cbb₃-2). These terminal oxidases are differentially regulated under various growth conditions and are thought to contribute to the survival of this microorganism in a wide variety of environmental niches. Here, we constructed multiple mutant strains of P. aeruginosa that express only one aerobic terminal oxidase to investigate the enzymatic characteristics and in vivo function of each enzyme. The K_m values of Cyo, CIO, and aa₃ for oxygen were similar and were 1 order of magnitude higher than those of cbb₃-1 and cbb₃-2, indicating that Cyo, CIO, and aa₃ are low-affinity enzymes and that cbb₃-1 and cbb₃-2 are high-affinity enzymes. Although cbb₃-1 and cbb₃-2 exhibited different expression patterns in response to oxygen concentration, they had similar K_m values for oxygen. Both cbb₃-1 and cbb₃-2 utilized cytochrome c₄ as the main electron donor under normal growth conditions. The electron transport chains terminated by cbb₃-1 and cbb₃-2 generate a proton gradient across the cell membrane with similar efficiencies. The electron transport chain of aa₃ had the highest proton translocation efficiency, whereas that of CIO had the lowest efficiency. The enzymatic properties of the terminal oxidases reported here are partially in agreement with their regulatory patterns and may explain the environmental adaptability and versatility of P. aeruginosa.     

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.     

Accommodation of NO in the active site of mammalian and bacterial cytochrome c oxidase aa3

Following different reports on the stoichiometry and configuration of NO binding to mammalian and bacterial reduced cytochrome c oxidase aa₃ (CcO), we investigated NO binding and dynamics in the active site of beef heart CcO as a function of NO concentration, using ultrafast transient absorption and EPR spectroscopy. We find that in the physiological range only one NO molecule binds to heme a₃, and time-resolved experiments indicate that even transient binding to Cu_B does not occur. Only at very high (∼ 2 mM) concentrations a second NO is accommodated in the active site, although in a different configuration than previously observed for CcO from Paracoccus denitrificans [E. Pilet, W. Nitschke, F. Rappaport, T. Soulimane, J.-C. Lambry, U. Liebl and M.H. Vos. Biochemistry 43 (2004) 14118-14127], where we proposed that a second NO does bind to Cu_B. In addition, in the bacterial enzyme two NO molecules can bind already at NO concentrations of ∼ 1 μM. The unexpected differences highlighted in this study may relate to differences in the physiological relevance of the CcO-NO interactions in both species.     

Structural and functional analysis of aa3-type and cbb3-type cytochrome c oxidases of Paracoccus denitrificans reveals significant differences in proton-pump design

In Paracoccusdenitrificans the aa₃-type cytochrome c oxidase and the bb₃-type quinol oxidase have previously been characterized in detail, both biochemically and genetically. Here we report on the isolation of a genomic locus that harbours the gene cluster ccoNOQP, and demonstrate that it encodes an alternative cbb₃-type cytochrome c oxidase. This oxidase has previously been shown to be specifically induced at low oxygen tensions, suggesting that its expression is controlled by an oxygen-sensing mechanism. This view is corroborated by the observation that the ccoNOQP gene cluster is preceded by a gene that encodes an FNR homologue and that its promoter region contains an FNR-binding motif. Biochemical and physiological analyses of a set of oxidase mutants revealed that, at least under the conditions tested, cytochromes aa₃, bb₃. and cbb₃ make up the complete set of terminal oxidases in P. denitrificans. Proton-translocation measurements of these oxidase mutants indicate that all three oxidase types have the capacity to pump protons. Previously, however, we have reported decreased H⁺/e coupling efficiencies of the cbb₃-type     

Operation of the cbb3-type terminal oxidase in Azotobacter vinelandii

A part of the gene encoding cbb ₃-type cytochrome oxidase CcoN subunit was cloned from Azotobacter vinelandii and a mutant strain of this bacterium with disrupted ccoN gene was constructed. In contrast to the wild type strain, this one is unable to oxidize cytochromes c ₄ and c ₅. Thus, the A. vinelandii respiratory chain is shown to contain cbb ₃-type cytochrome c oxidase. It is also shown that the activity of this enzyme is not necessary for diazotrophic growth of A. vinelandii at high oxygen concentrations.     

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 KC Channel in the cbb3-Type Respiratory Oxygen Reductase from Rhodobacter capsulatus Is Required for Both Chemical and Pumped Protons

The heme-copper superfamily of proton-pumping respiratory oxygen reductases are classified into three families (A, B, and C families) based on structural and phylogenetic analyses. Most studies have focused on the A family, which includes the eukaryotic mitochondrial cytochrome c oxidase as well as many bacterial homologues. Members of the C family, also called the cbb₃-type oxygen reductases, are found only in prokaryotes and are of particular interest because of their presence in a number of human pathogens. All of the heme-copper oxygen reductases require proton-conducting channels to convey chemical protons to the active site for water formation and to convey pumped protons across the membrane. Previous work indicated that there is only one proton-conducting input channel (the K^C channel) present in the cbb₃-type oxygen reductases, which, if correct, must be utilized by both chemical protons and pumped protons. In this work, the effects of mutations in the K^C channel of the cbb₃-type oxygen reductase from Rhodobacter capsulatus were investigated by expressing the mutants in a strain lacking other respiratory oxygen reductases. Proton pumping was evaluated by using intact cells, and catalytic oxygen reductase activity was measured in isolated membranes. Two mutations, N346M and Y374F, severely reduced catalytic activity, presumably by blocking the chemical protons required at the active site. One mutation, T272A, resulted in a substantially lower proton-pumping stoichiometry but did not inhibit oxygen reductase activity. These are the first experimental data in support of the postulate that pumped protons are taken up from the bacterial cytoplasm through the K^C channel.     

Isolation and Characterization of Rhodobacter capsulatus Mutants Affected in Cytochrome cbb3 Oxidase Activity

The facultative phototrophic bacterium Rhodobacter capsulatus contains only one form of cytochrome (cyt) c oxidase, which has recently been identified as a cbb₃-type cyt c oxidase. This is unlike other related species, such as Rhodobacter sphaeroides and Paracoccus denitrificans, which contain an additional mitochondrial-like aa₃-type cyt c oxidase. An extensive search for mutants affected in cyt c oxidase activity in R. capsulatus led to the isolation of at least five classes of mutants. Plasmids complementing them to a wild-type phenotype were obtained for all but one of these classes from a chromosomal DNA library. The first class of mutants contained mutations within the structural genes (ccoNOQP) of the cyt cbb₃ oxidase. Sequence analysis of these mutants and of the plasmids complementing them revealed that ccoNOQP in R. capsulatus is not flanked by the oxygen response regulator fnr, which is located upstream of these genes in other species. Genetic and biochemical characterizations of mutants belonging to this group indicated that the subunits CcoN, CcoO, and CcoP are required for the presence of an active cyt cbb₃ oxidase, and unlike in Bradyrhizobium japonicum, no active CcoN-CcoO subcomplex was found in R. capsulatus. In addition, mutagenesis experiments indicated that the highly conserved open reading frame 277 located adjacent to ccoNOQP is required neither for cyt cbb₃ oxidase activity or assembly nor for respiratory or photosynthetic energy transduction in R. capsulatus. The remaining cyt c oxidase-minus mutants mapped outside of ccoNOQP and formed four additional groups. In one of these groups, a fully assembled but inactive cyt cbb₃ oxidase was found, while another group had only extremely small amounts of it. The next group was characterized by a pleiotropic effect on all membrane-bound c-type cytochromes, and the remaining mutants not complemented by the plasmids complementing the first four groups formed at least one additional group affecting the biogenesis of the cyt cbb₃ oxidase of R. capsulatus.The gram-negative facultative photosynthetic bacterium Rhodobacter capsulatus has a highly branched electron transport chain, resulting in its ability to grow under a wide variety of conditions (52). Its light-driven photosynthetic electron transfer pathway is a cyclic process between the photochemical reaction center and the ubihydroquinone cytochrome (cyt) c oxidoreductase (cyt bc₁ complex) (30). On the other hand, the respiratory electron transfer pathways of R. capsulatus are branched after the quinone pool and contain two different terminal oxidases, previously called cyt b₄₁₀ (cyt c oxidase) and cyt b₂₆₀ (quinol oxidase) (3, 27, 29, 53). The branch involving cyt c oxidase is similar to the mitochondrial electron transfer chain in that it depends on the cyt bc₁ complex and a c-type cyt acting as an electron carrier. The quinol oxidase branch circumvents the cyt bc₁ complex and the cyt c oxidase by taking electrons directly from the quinone pool to reduce O₂ to H₂O. The pronounced metabolic versatility, including the ability to grow under dark, anaerobic conditions (50, 52), makes these purple non-sulfur bacteria excellent model organisms for studying microbial energy transduction.Marrs and Gest (29) have reported the first R. capsulatus mutants which were defective in the respiratory electron transport chain. Of these mutants, M5 was incapable of catalyzing the α-naphthol plus N′,N′-dimethyl-p-phenylenediamine (DMPD) plus O₂→indophenol blue plus H₂O reaction (NADI reaction) and unable to grow by respiration (Res⁻), and hence was deficient in both terminal oxidases. Another mutant, M4, was also NADI⁻ but Res⁺ due to the presence of an active quinol oxidase. Marrs and Gest have also described two different spontaneous revertants of M5, called M6 and M7, which regained the ability to grow by respiration (29). M6 regained cyt c oxidase activity and became concurrently NADI⁺ and sensitive to low concentrations of cyanide and the cyt bc₁ inhibitor myxothiazol, but remained quinol oxidase⁻. On the other hand, M7 regained the quinol oxidase activity but remained cyt c oxidase⁻ (thus, NADI⁻ and resistant to myxothiazol, a phenotype identical to that of M4). All of these mutants remained proficient for phototrophic (Ps) growth.The cyt c oxidase of R. capsulatus has been purified previously and characterized as being a novel cbb₃-type cyt c oxidase without a Cu_A center (15). It is composed of at least a membrane-integral b-type cyt (subunit I [CcoN]) with a low-spin heme b and a high-spin heme b₃-Cu_B binuclear center, and two membrane-anchored c-type cyts (CcoO and CcoP). It has a unique active site that possibly confers a very high affinity for its substrate oxygen (49). The structural genes of this enzyme (ccoNOQP) have been sequenced recently from R. capsulatus 37b4 (45) and aligned to the partial amino acid sequence of the purified enzyme from R. capsulatus MT1131 (15). Although a ccoN mutant of strain 37b4 was reported to lack cyt c oxidase activity (45), the observed discrepancies between the amino acid sequence and the nucleotide sequence do not entirely exclude the possible presence of two similar cb-type cyt c oxidases in this species. The presence of a similar cyt c oxidase has also been demonstrated in several other bacteria, including P. denitrificans (9), R. sphaeroides (13), and Rhizobium spp. In the latter species, the homologs of ccoNOQP have been named fixNOQP (23, 34) and are required to support respiration under oxygen-limited growth during symbiotic nitrogen fixation (36).The biogenesis of a multisubunit protein complex containing several prosthetic groups, such as cyt cbb₃ oxidase, is likely to require many accessory proteins involved in various posttranslational events, including protein translocation, assembly, cofactor insertion, and maturation (46). Thus, insights into this important biological process, about which currently little is known, may be gained by searching for mutants defective in cyt c oxidase activity. In this work, we describe the isolation of such mutants and their molecular genetic characterization, including those already available, such as M4, M5, and M7G. These studies indicate that in R. capsulatus, gene products of at least five different loci are involved in the formation of an active cyt cbb₃ oxidase.     

Active Site of Cytochrome cbb3

Cytochrome cbb₃ is the most distant member of the heme-copper oxidase family still retaining the following major feature typical of these enzymes: reduction of molecular oxygen to water coupled to proton translocation across the membrane. The thermodynamic properties of the six redox centers, five hemes and a copper ion, in cytochrome cbb₃ from Rhodobacter sphaeroides were studied using optical and EPR spectroscopy. The low spin heme b in the catalytic subunit was shown to have the highest midpoint redox potential (E_m,7 +418 mV), whereas the three hemes c in the two other subunits titrated with apparent midpoint redox potentials of +351, +320, and +234 mV. The active site high spin heme b₃ has a very low potential (E_m,7 -59 mV) as opposed to the copper center (Cu_B), which has a high potential (E_m,7 +330 mV). The EPR spectrum of the ferric heme b₃ has rhombic symmetry. To explain the origins of the rhombicity, the Glu-383 residue located on the proximal side of heme b₃ was mutated to aspartate and to glutamine. The latter mutation caused a 10 nm blue shift in the optical reduced minus oxidized heme b₃ spectrum, and a dramatic change of the EPR signal toward more axial symmetry, whereas mutation to aspartate had far less severe consequences. These results strongly suggest that Glu-383 is involved in hydrogen bonding to the proximal His-405 ligand of heme b₃, a unique interaction among heme-copper oxidases.The heme-copper oxidases form a family of enzymes that have structural homology of the catalytic subunit in common (1). This family of proteins, characterized by six conserved histidine ligands of the redox cofactors, ranges from classical, mitochondrial terminal oxidases to nitric-oxide reductases, and the members have been classified according to evolutionary relationships of their sequences (2–4). The bacterial cbb₃-type cytochrome c oxidases form a distinct, divergent subfamily within the heme-copper oxidases (5). Terminal oxidases share the catalytic activity of four-electron reduction of molecular oxygen to water coupled to translocation of protons across the membrane (6, 7). Cytochrome cbb₃, expressed in some bacteria as a sole terminal oxidase, is characterized by its ability to maintain catalytic activity under low oxygen tension (8), and it has also been shown to have the capacity to translocate protons (9).The Rhodobacter sphaeroides cytochrome cbb₃ is encoded by the ccoNOQP operon composed of four genes (10). The catalytic subunit CcoN homes a binuclear active site composed of a high spin heme b₃ and a nearby copper ion (Cu_B). There are altogether four low spin hemes in the enzyme. In addition to a protoheme (heme b) residing in the vicinity of the active site in subunit CcoN, there are three hemes c present in the soluble domains of the two other transmembrane subunits, a monoheme subunit CcoO and a diheme subunit CcoP (11). There is yet one more membrane-spanning subunit, CcoQ, without bound cofactors (12). Although the catalytic subunit shows homology to the other heme-copper oxidases (13), the other three subunits bear no resemblance to subunits of other types of terminal oxidases. However, subunit CcoO has been shown to have sequence homology with the nitric-oxide reductase subunit NorC (14).The crystal structures of a few heme-copper oxidases have been resolved (15–19), but only structural homology models are currently available for cytochromes cbb₃ (20–23). Apart from the signatures common to all heme-copper oxidases, the sequence alignments have revealed only very few other conserved residues when terminal oxidases are compared. Even though some amino acids, absent from cytochrome cbb₃, have been shown to be of critical importance to the function of the classical heme-copper oxidases, the major functions still remain the same in all of these enzymes.The thermodynamic properties of the cbb₃-type oxidases have been investigated sparsely. Apart from work yielding partial information about the properties of the hemes (11, 24, 25), two more complete studies have been carried out (5, 26). All the hemes in cytochrome cbb₃ were proposed to have high redox potentials, both in the Pseudomonas stutzeri and Bradyrhizobium japonicum enzymes (5, 26). This is also the case in all other studies, except for the enzyme from Rhodothermus marinus, where two low potential redox centers were reported (25). However, little is known about the copper center in the active site (Cu_B). Early Fourier transform infrared (FTIR)² spectroscopic measurements identified the presence of a heme/copper binuclear center in R. sphaeroides cytochrome cbb₃ (11), and more recent resonance Raman and FTIR studies have given additional information about the structure of the active site (27–29).In the absence of deconvoluted spectral components and thereby clear assignments of the redox centers in the cbb₃-type oxidases, and the lack of consensus about their thermodynamic properties, a complete study was required. In this work we have set out to investigate the thermodynamic properties of all the redox centers in cytochrome cbb₃ from R. sphaeroides using a combination of optical and EPR redox titrations with the main focus on the details of the catalytic site. This effort will form a basis for further mechanistic studies.