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.     

Structural Alterations in a Component of Cytochrome c Oxidase and Molecular Evolution of Pathogenic Neisseria in Humans

Three closely related bacterial species within the genus Neisseria are of importance to human disease and health. Neisseria meningitidis is a major cause of meningitis, while Neisseria gonorrhoeae is the agent of the sexually transmitted disease gonorrhea and Neisseria lactamica is a common, harmless commensal of children. Comparative genomics have yet to yield clear insights into which factors dictate the unique host-parasite relationships exhibited by each since, as a group, they display remarkable conservation at the levels of nucleotide sequence, gene content and synteny. Here, we discovered two rare alterations in the gene encoding the CcoP protein component of cytochrome cbb ₃ oxidase that are phylogenetically informative. One is a single nucleotide polymorphism resulting in CcoP truncation that acts as a molecular signature for the species N. meningitidis. We go on to show that the ancestral ccoP gene arose by a unique gene duplication and fusion event and is specifically and completely distributed within species of the genus Neisseria. Surprisingly, we found that strains engineered to express either of the two CcoP forms conditionally differed in their capacity to support nitrite-dependent, microaerobic growth mediated by NirK, a nitrite reductase. Thus, we propose that changes in CcoP domain architecture and ensuing alterations in function are key traits in successive, adaptive radiations within these metapopulations. These findings provide a dramatic example of how rare changes in core metabolic proteins can be connected to significant macroevolutionary shifts. They also show how evolutionary change at the molecular level can be linked to metabolic innovation and its reversal as well as demonstrating how genotype can be used to infer alterations of the fitness landscape within a single host.     

Characterization of an ntrX Mutant of Neisseria gonorrhoeae Reveals a Response Regulator That Controls Expression of Respiratory Enzymes in Oxidase-Positive Proteobacteria

NtrYX is a sensor-histidine kinase/response regulator two-component system that has had limited characterization in a small number of Alphaproteobacteria. Phylogenetic analysis of the response regulator NtrX showed that this two-component system is extensively distributed across the bacterial domain, and it is present in a variety of Betaproteobacteria, including the human pathogen Neisseria gonorrhoeae. Microarray analysis revealed that the expression of several components of the respiratory chain was reduced in an N. gonorrhoeae ntrX mutant compared to that in the isogenic wild-type (WT) strain 1291. These included the cytochrome c oxidase subunit (ccoP), nitrite reductase (aniA), and nitric oxide reductase (norB). Enzyme activity assays showed decreased cytochrome oxidase and nitrite reductase activities in the ntrX mutant, consistent with microarray data. N. gonorrhoeae ntrX mutants had reduced capacity to survive inside primary cervical cells compared to the wild type, and although they retained the ability to form a biofilm, they exhibited reduced survival within the biofilm compared to wild-type cells, as indicated by LIVE/DEAD staining. Analyses of an ntrX mutant in a representative alphaproteobacterium, Rhodobacter capsulatus, showed that cytochrome oxidase activity was also reduced compared to that in the wild-type strain SB1003. Taken together, these data provide evidence that the NtrYX two-component system may be a key regulator in the expression of respiratory enzymes and, in particular, cytochrome c oxidase, across a wide range of proteobacteria, including a variety of bacterial pathogens.     

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.     

Cytochrome c‐based domain modularity governs genus‐level diversification of electron transfer to dissimilatory nitrite reduction

The genus Neisseria contains two pathogenic species (N. meningitidis and N. gonorrhoeae) in addition to a number of commensal species that primarily colonize mucosal surfaces in man. Within the genus, there is considerable diversity and apparent redundancy in the components involved in respiration. Here, we identify a unique c‐type cytochrome (c_N) that is broadly distributed among commensal Neisseria, but absent in the pathogenic species. Specifically, c_N supports nitrite reduction in N. gonorrhoeae strains lacking the cytochromes c₅ and CcoP established to be critical to NirK nitrite reductase activity. The c‐type cytochrome domain of c_N shares high sequence identity with those localized c‐terminally in c₅ and CcoP and all three domains were shown to donate electrons directly to NirK. Thus, we identify three distinct but paralogous proteins that donate electrons to NirK. We also demonstrate functionality for a N. weaverii NirK variant with a C‐terminal c‐type heme extension. Taken together, modular domain distribution and gene rearrangement events related to these respiratory electron carriers within Neisseria are concordant with major transitions in the macroevolutionary history of the genus. This work emphasizes the importance of denitrification as a selectable trait that may influence speciation and adaptive diversification within this largely host‐restricted bacterial genus.     

A Biochemical Approach to Study the Role of the Terminal Oxidases in Aerobic Respiration in Shewanella oneidensis MR-1

The genome of the facultative anaerobic γ-proteobacterium Shewanella oneidensis MR-1 encodes for three terminal oxidases: a bd-type quinol oxidase and two heme-copper oxidases, a A-type cytochrome c oxidase and a cbb ₃-type oxidase. In this study, we used a biochemical approach and directly measured oxidase activities coupled to mass-spectrometry analysis to investigate the physiological role of the three terminal oxidases under aerobic and microaerobic conditions. Our data revealed that the cbb ₃-type oxidase is the major terminal oxidase under aerobic conditions while both cbb ₃-type and bd-type oxidases are involved in respiration at low-O₂ tensions. On the contrary, the low O₂-affinity A-type cytochrome c oxidase was not detected in our experimental conditions even under aerobic conditions and would therefore not be required for aerobic respiration in S. oneidensis MR-1. In addition, the deduced amino acid sequence suggests that the A-type cytochrome c oxidase is a ccaa ₃-type oxidase since an uncommon extra-C terminal domain contains two c-type heme binding motifs. The particularity of the aerobic respiratory pathway and the physiological implication of the presence of a ccaa ₃-type oxidase in S. oneidensis MR-1 are discussed.     

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.     

The ccoNOQP gene cluster codes for a cb-type cytochrome oxidase that functions in aerobic respiration of Rhodobacter capsulatus

The genes for a new type of a haem-copper cytochrome oxidase were cloned from Rhodobacter capsulatus strain 37b4, using the Bradyrhizobium japonicum fixNOQP gene region as a hybridizing probe. Four genes, probably organized in an operon (ccoNOQP), were identified; their products share extensive amino acid sequence similarity with the FixN, O, Q and P proteins that have recently been shown to be the subunits of a cb-type oxidase. CcoN is a c-type cytochrome, CcoO and CcoP are membrane-bound mono- and dihaem c-type cytochromes and CcoQ is a small membrane protein of unknown function. Genes for a similar oxidase are also present in other non-rhizobial bacterial species such as Azoto-bacter vinelandii, Agrobacterium tumefaciens and Pseudomonas aeruginosa, as revealed by polymerase chain reaction analysis. A ccoN mutant was constructed whose phenotype, in combination with the structural information on the gene products, provides evidence that the CcoNOQP oxidase is a cytochrome c oxidase of the cb type, which supports aerobic respiration in R. capsulatus and which is probably identical to the cbb₃-type oxidase that was recently purified from a different strain of the same species. Mutant analysis also showed that this oxidase has no influence on photosynthetic growth and nitrogen-fixation activity.     

Biochemical and Biophysical Characterization of the Two Isoforms of cbb3-Type Cytochrome c Oxidase from Pseudomonas stutzeri

The cbb₃-type cytochrome c oxidases (cbb₃-CcOs) are members of the heme-copper oxidase superfamily that couple the reduction of oxygen to translocation of protons across the membrane. The cbb₃-CcOs are present only in bacteria and play a primary role in microaerobic respiration, being essential for nitrogen-fixing endosymbionts and for some human pathogens. As frequently observed in Pseudomonads, Pseudomonas stutzeri contains two independent ccoNO(Q)P operons encoding the two cbb₃ isoforms, Cbb₃-1 and Cbb₃-2. While the crystal structure of Cbb₃-1 from P. stutzeri was determined recently and cbb₃-CcOs from other organisms were characterized functionally, less emphasis has been placed on the isoform-specific differences between the cbb₃-CcOs. In this work, both isoforms were homologously expressed in P. stutzeri strains from which the genomic version of the respective operon was deleted. We purified both cbb₃ isoforms separately by affinity chromatography and increased the yield of Cbb₃-2 to a similar level as Cbb₃-1 by replacing its native promoter. Mass spectrometry, UV-visible (UV-Vis) spectroscopy, differential scanning calorimetry, as well as oxygen reductase and catalase activity measurements were employed to characterize both cbb₃ isoforms. Differences were found concerning the thermal stability and the presence of subunit CcoQ. However, no significant differences between the two isoforms were observed otherwise. Interestingly, a surprisingly high turnover of at least 2,000 electrons s⁻¹ and a high Michaelis-Menten constant (K_m ∼ 3.6 mM) using ascorbate–N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride (TMPD) as the electron donor were characteristic for both P. stutzeri cbb₃-CcOs. Our work provides the basis for further mutagenesis studies of each of the two cbb₃ isoforms specifically.     

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.     

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.     

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.     

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.     

Genetic evidence for 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) as a negative effector of cytochrome terminal oxidase cbb 3 production in Rhizobium etli

A Rhizobium etli Tn5mob-induced mutant (CFN035) exhibits an enhanced capacity to oxidize N,N,N′,N′, tetramethyl-p -phenylenediamine (TMPD), a presumptive indicator of elevated cytochrome c terminal oxidase activity. Sequencing of the mutated gene in CFN035 revealed that it codes for the amidophosphoribosyl transferase enzyme (PurF) that catalyzes the first step in the purine biosynthetic pathway. Two c-type cytochromes with molecular weights of 32 and 27?kDa were produced in strain CFN035, which also produced a novel CO-reactive cytochrome (absorbance trough at 553?nm), in contrast to strain CE3 which produced a single 32?kDa c-type protein and did not produce the 553?nm CO-reactive cytochrome. A wild-type R. etli strain that expresses the Bradyrhizobium japonicum fixNOQP genes, which code for the symbiotic cytochrome terminal oxidase cbb ₃, produced similar absorbance spectra (a trough at 553?nm in CO-difference spectra) and two c -type proteins similar in size to those of strain CFN035, suggesting that CFN035 also produces the cbb ₃ terminal oxidase. The expression of a R. etli fixN-lacZ gene fusion was measured in several R. etli mutants affected in different steps of the purine biosynthetic pathway. Our analysis showed that purF, purD, purQ, purL, purY, purK and purE mutants expressed three-fold higher levels of the fixNOQP operon than the wild-type strain. The derepressed expression of fixN was not observed in a purH mutant. The purH gene product catalyzes the conversion of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) and inosine. Supplementation with AICA riboside lowered the levels of fixN expression in the purF mutants. These data are consistent with the possibility that AICAR, or a closely related metabolite, is a negative effector of the production of the symbiotic terminal oxidase cbb ₃ in R. etli.     

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.     

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     

Lumenal proteins involved in respiratory electron transport in the cyanobacterium Synechocystis sp. PCC6803

Cyanobacterial thylakoids catalyze both photosynthetic and respiratory activities. In a photosystem I-less Synechocystis sp. PCC 6803 strain, electrons generated by photosystem II appear to be utilized by cytochrome oxidase. To identify the lumenal electron carriers (plastocyanin and/or cytochromes c ₅₅₃, c ₅₅₀, and possibly c _M) that are involved in transfer of photosystem II-generated electrons to the terminal oxidase, deletion constructs for genes coding for these components were introduced into a photosystem I-less Synechocystis sp. PCC 6803 strain, and electron flow out of photosystem II was monitored in resulting strains through chlorophyll fluorescence yields. Loss of cytochrome c ₅₅₃ or plastocyanin, but not of cytochrome c ₅₅₀, decreased the rate of electron flow out of photosystem II. Surprisingly, cytochrome c _M could not be deleted in a photosystem I-less background strain, and also a double-deletion mutant lacking both plastocyanin and cytochromec ₅₅₃ could not be obtained. Cytochrome c _M has some homology with the cytochrome c-binding regions of the cytochromecaa₃ -type cytochrome oxidase from Bacillus spp. and Thermus thermophilus. We suggest that cytochrome c _M is a component of cytochrome oxidase in cyanobacteria that serves as redox intermediate between soluble electron carriers and the cytochromeaa₃ complex, and that either plastocyanin or cytochrome c ₅₅₃ can shuttle electrons from the cytochrome b_6f complex to cytochrome c _M.     

Transient Binding of CO to CuB in Cytochrome c Oxidase Is Dynamically Linked to Structural Changes around a Carboxyl Group: A Time-Resolved Step-Scan Fourier Transform Infrared Investigation

The redox-driven proton pump cytochrome c oxidase is that enzymatic machinery of the respiratory chain that transfers electrons from cytochrome c to molecular oxygen and thereby splits molecular oxygen to form water. To investigate the reaction mechanism of cytochrome c oxidase on the single vibrational level, we used time-resolved step-scan Fourier transform infrared spectroscopy and studied the dynamics of the reduced enzyme after photodissociation of bound carbon monoxide across the midinfrared range (2300-950 cm⁻¹). Difference spectra of the bovine complex were obtained at -20°C with 5 μs time resolution. The data demonstrate a dynamic link between the transient binding of CO to Cu_B and changes in hydrogen bonding at the functionally important residue E(I-286). Variation of the pH revealed that the pK_a of E(I-286) is >9.3 in the fully reduced CO-bound oxidase. Difference spectra of cytochrome c oxidase from beef heart are compared with those of the oxidase isolated from Rhodobacter sphaeroides. The bacterial enzyme does not show the environmental change in the vicinity of E(I-286) upon CO dissociation. The characteristic band shape appears, however, in redox-induced difference spectra of the bacterial enzyme but is absent in redox-induced difference spectra of mammalian enzyme. In conclusion, it is demonstrated that the dynamics of a large protein complex such as cytochrome c oxidase can be resolved on the single vibrational level with microsecond Fourier transform infrared spectroscopy. The applied methodology provides the basis for future investigations of the physiological reaction steps of this important enzyme.