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.     

Mutagenesis of tyrosine residues within helix VII in subunit I of the cytochrome <Emphasis Type="Italic">cbb</Emphasis><Subscript>3</Subscript> oxidase from <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis>

The cbb ₃-type oxidases are members of the heme-copper oxidase superfamily, distant by sequence comparisons, but sharing common functional characteristics. The cbb ₃ oxidases are missing an active-site tyrosine residue that is absolutely conserved in all A and B-type heme-copper oxidases. This tyrosine is known to play a critical role in the catalytic mechanisms of A and B-type oxidases. The absence of this tyrosine in the cbb ₃ oxidases raises the possibility that the cbb ₃ oxidases utilize a different catalytic mechanism from that of the other members of the superfamily, or have this conserved residue in different helices. Recently sequence comparisons indicate that, a tyrosine residues that might be analogous to the active-site tyrosine in other oxidases are present in the cbb ₃ oxidases but these tyrosines originates from a different transmembrane helix within the protein. In this research, three conserved tyrosine residues, Y294, Y308 and Y318, in helix VII were substituted for phenylalanine. Y318F mutant in the Rhodobacter capsulatus oxidase resulted in a fully assembled enzyme with nativelike structure and activity, but Y294F mutant is not assembled and have a catalytic activity. On the other hand, Y308F mutant is fully assembled enzyme with nativelike structure, but lacking catalytic activity. This result indicates that Y308 should be crucial in catalytic activity of the cbb ₃ oxidase of R. capsulatus. These findings support the assumption that all of the heme-copper oxidases utilize the same catalytic mechanism and provide a residue originates from different places within the primary sequence for different members of the same superfamily.     

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.     

Regulation of Metabolic and Electron Transport Pathways in the Freshwater Bacterium Beggiatoa leptomitiformis D-402

The biomass yield of freshwater filamentous sulfur bacteria of the genus Beggiatoa, when grown lithoheterotrophically or mixotrophically, has been shown to increase 2 to 2.5 times under microaerobic conditions (0.12 mg/l oxygen) as compared to aerobic conditions (9 mg/l oxygen). The activity of the glyoxylate cycle key enzymes have been found to increase two to three times under microaerobic conditions (at an O₂ concentration of 2 mg/l), and the activities of the sulfur metabolism enzymes increased three to five times (at an O₂ concentration of 0.1–0.5 mg/l). It has also been found that, under microaerobic conditions, thiosulfate was almost completely oxidized to sulfate by the bacteria, without accumulation of intermediate metabolites. At the same time, a 2- to 15-fold decrease in the activities of the tricarboxylic acid cycle enzymes involved in the reduction of NAD and FAD was observed. Reorganization of the respiratory chain after changes in aeration and type of nutrition was also observed. It has been found that, in cells grown heterotrophically, the terminal part of the respiratory chain contained an aa ₃-type oxidase, whereas, during mixotrophic, lithoheterotrophic, and autotrophic growth, aa ₃-type oxidase synthesis was inhibited, and the synthesis of a cbb ₃-type oxidase, which is induced under microaerobic conditions, was activated. The gene of the catalytic subunit CcoN of the cbb ₃-type oxidase was sequenced and proved to be highly homologous to the corresponding genes of other proteobacteria.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 452–459.Original Russian Text Copyright © 2005 by Muntyan, Grabovich, Patritskaya, Dubinina.     

Electron and proton transfer in the <Emphasis Type="Italic">ba</Emphasis><Subscript>3</Subscript> oxidase from <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

The ba ₃-type cytochrome c oxidase from Thermus thermophilus is phylogenetically very distant from the aa ₃–type cytochrome c oxidases. Nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of O₂ to water is catalysed at a haem a ₃-Cu_B catalytic site. The three-dimensional structure of the ba ₃ oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial, Rhodobacter sphaeroides and Paracoccus denitrificans aa ₃ oxidases. In this study we investigated the oxidative part of the catalytic cycle of the ba ₃ -cytochrome c oxidase using the flow-flash method. After flash-induced dissociation of CO from the fully reduced enzyme in the presence of oxygen we observed rapid oxidation of cytochrome b (k ≅ 6.8 × 10⁴ s⁻¹) and formation of the peroxy (P_R) intermediate. In the next step a proton was taken up from solution with a rate constant of ~1.7 × 10⁴ s⁻¹, associated with formation of the ferryl (F) intermediate, simultaneous with transient reduction of haem b. Finally, the enzyme was oxidized with a rate constant of ~1,100 s⁻¹, accompanied by additional proton uptake. The total proton uptake stoichiometry in the oxidative part of the catalytic cycle was ~1.5 protons per enzyme molecule. The results support the earlier proposal that the P_R and F intermediate spectra are similar (Siletsky et al. Biochim Biophys Acta 1767:138, 2007) and show that even though the architecture of the proton-conducting pathways is different in the ba ₃ oxidases, the proton-uptake reactions occur over the same time scales as in the aa ₃-type oxidases. Smirnova and Zaslavsky contributed equally to the work described in this paper.     

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.     

A decade of crystallization drops: Crystallization of the cbb3 cytochrome c oxidase from Pseudomonas stutzeri

The cbb₃ cytochrome c oxidases are distant members of the superfamily of heme copper oxidases. These terminal oxidases couple O₂ reduction with proton transport across the plasma membrane and, as a part of the respiratory chain, contribute to the generation of an electrochemical proton gradient. Compared with other structurally characterized members of the heme copper oxidases, the recently determined cbb₃ oxidase structure at 3.2 Å resolution revealed significant differences in the electron supply system, the proton conducting pathways and the coupling of O₂ reduction to proton translocation. In this paper, we present a detailed report on the key steps for structure determination. Improvement of the protein quality was achieved by optimization of the number of lipids attached to the protein as well as the separation of two cbb₃ oxidase isoenzymes. The exchange of n‐dodecyl‐β‐d ‐maltoside for a precisely defined mixture of two α‐maltosides and decanoylsucrose as well as the choice of the crystallization method had a most profound impact on crystal quality. This report highlights problems frequently encountered in membrane protein crystallization and offers meaningful approaches to improve crystal quality.     

Combined effect of loss of the caa3 oxidase and Crp regulation drives Shewanella to thrive in redox-stratified environments

Shewanella species are a group of facultative Gram-negative microorganisms with remarkable respiration abilities that allow the use of a diverse array of terminal electron acceptors (EA). Like most bacteria, S. oneidensis possesses multiple terminal oxidases, including two heme-copper oxidases (caa₃- and cbb₃-type) and a bd-type quinol oxidase. As aerobic respiration is energetically favored, mechanisms underlying the fact that these microorganisms thrive in redox-stratified environments remain vastly unexplored. In this work, we discovered that the cbb₃-type oxidase is the predominant system for respiration of oxygen (O₂), especially when O₂ is abundant. Under microaerobic conditions, the bd-type quinol oxidase has a significant role in addition to the cbb₃-type oxidase. In contrast, multiple lines of evidence suggest that under test conditions the caa₃-type oxidase, an analog to the mitochondrial enzyme, has no physiological significance, likely because of its extremely low expression. In addition, expression of both cbb₃- and bd-type oxidases is under direct control of Crp (cAMP receptor protein) but not the well-established redox regulator Fnr (fumarate nitrate regulator) of canonical systems typified in Escherichia coli. These data, collectively, suggest that adaptation of S. oneidensis to redox-stratified environments is likely due to functional loss of the caa₃-type oxidase and switch of the regulatory system for respiration.     

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.     

Evolution of the Cytochrome c Oxidase Proton Pump

The superfamily of quinol and cytochrome c terminal oxidase complexes is related by a homologous subunit containing six positionally conserved histidines that ligate a low-spin heme and a heme–copper dioxygen activating and reduction center. On the basis of the structural similarities of these enzymes, it has been postulated that all members of this superfamily catalyze proton translocation by similar mechanisms and that the Cu_A center found in most cytochrome c oxidase complexes serves merely as an electron conduit shuttling electrons from ferrocytochrome c into the hydrophobic core of the enzyme. The recent characterization of cytochrome c oxidase complexes and structurally similar cytochrome c:nitric oxide oxidoreductase complexes without Cu_A centers has strengthened this view. However, recent experimental evidence has shown that there are two ubiquinone(ol) binding sites on the Escherichia coli cytochrome bo ₃ complex in dynamic equilibrium with the ubiquinone(ol) pool, thereby strengthening the argument for a Q(H₂)-loop mechanism of proton translocation [Musser SM et al. (1997) Biochemistry 36:894–902]. In addition, a number of reports suggest that a Q(H₂)-loop or another alternate proton translocation mechanism distinct from the mitochondrial aa ₃ -type proton pump functions in Sulfolobus acidocaldarius terminal oxidase complexes. The possibility that a primitive quinol oxidase complex evolved to yield two separate complexes, the cytochrome bc ₁ and cytochrome c oxidase complexes, is explored here. This idea is the basis for an evolutionary tree constructed using the notion that respiratory complexity and efficiency progressively increased throughout the evolutionary process. The analysis suggests that oxygenic respiration is quite an old process and, in fact, predates nitrogenic respiration as well as reaction-center photosynthesis. Received: 11 June 1997 / Accepted: 30 October 1997     

The regulation of cytochrome c oxidase of Rhodobacter capsulatus by light and oxygen

In order to distinguish between the regulatory effects of oxygen tension and light intensity on cytochrome c oxidase protein and enzymatic activity cells of Rhodobacter capsulatus were shifted from phototrophic (anaerobic, light) growth to aerobic-light, aerobic-dark and to anaerobic-dark conditions, respectively. During shift-experiments the formation of oxidase protein and regulation of oxidase activity was followed by immunological and enzymatic means. The results support the idea, that the formation of oxidase protein is regulated by oxygen tension and light intensity changes, whereas the regulation of oxidase activity seems only to be correlated to the oxygen tension. A DNA sequence involved in the oxygen-dependent regulation of cytochrome oxidase could be identified in the regulation-deficient oxidase mutant H41 of R. capsulatus. Immunological investigations of cytochrome c ₂ from mutant H41 demonstrated at the same time the participation of the c ₂-polypeptide in the regulation of cytochrome c oxidase.Abbreviations Bchl bacteriochlorophyll - CIE crossed immuno-electrophoresis - DMSO dimethyl sulfoxide     

Structure and coordination of CuB in the Acidianus ambivalens aa 3 quinol oxidase heme–copper center

The coordination environment of the Cu_B center of the quinol oxidase from Acidianus ambivalens, a type B heme–copper oxygen reductase, was investigated by Fourier transform (FT) IR and extended X-ray absorption fine structure (EXAFS) spectroscopy. The comparative structural chemistry of dinuclear Fe–Cu sites of the different types of oxygen reductases is of great interest. Fully reduced A. ambivalens quinol oxidase binds CO at the heme a ₃ center, with ν(CO)=1,973 cm⁻¹. On photolysis, the CO migrated to the Cu_B center, forming a Cu_B^I–CO complex with ν(CO)=2,047 cm⁻¹. Raising the temperature of the samples to 25°C did not result in a total loss of signal in the FTIR difference spectrum although the intensity of these signals was reduced sevenfold. This observation is consistent with a large energy barrier against the geminate rebinding of CO to the heme iron from Cu_B, a restricted limited access at the active-site pocket for a second binding, and a kinetically stable Cu_B–CO complex in A. ambivalens aa ₃. The Cu_B center was probed in a number of different states using EXAFS spectroscopy. The oxidized state was best simulated by three histidines and a solvent O scatterer. On reduction, the site became three-coordinate, but in contrast to the bo ₃ enzyme, there was no evidence for heterogeneity of binding of the coordinated histidines. The Cu_B centers in both the oxidized and the reduced enzymes also appeared to contain substoichiometric amounts (0.2 mol equiv) of nonlabile chloride ion. EXAFS data of the reduced carbonylated enzyme showed no difference between dark and photolyzed forms. The spectra could be well fit by 2.5 imidazoles, 0.5 Cl⁻ and 0.5 CO ligands. This arrangement of scatterers would be consistent with about half the sites remaining as unligated Cu(his)₃ and half being converted to Cu(his)₂Cl⁻CO, a 50/50 ratio of Cu(his)₂Cl⁻ and Cu(his)₃CO, or some combination of these formulations. Electronic Supplementary Material Supplementary material is available for this article at .     

Thebc 1 complexes ofRhodobacter sphaeroides andRhodobacter capsulatus

Photosynthetic bacteria offer excellent experimental opportunities to explore both the structure and function of the ubiquinol-cytochromec oxidoreductase (bc ₁ complex). In bothRhodobacter sphaeroides andRhodobacter capsulatus, thebc ₁ complex functions in both the aerobic respiratory chain and as an essential component of the photosynthetic electron transport chain. Because thebc ₁ complex in these organisms can be functionally coupled to the photosynthetic reaction center, flash photolysis can be used to study electron flow through the enzyme and to examine the effects of various amino acid substitutions. During the past several years, numerous mutations have been generated in the cytochromeb subunit, in the Rieske iron-sulfur subunit, and in the cytochromec ₁ subunit. Both site-directed and random mutagenesis procedures have been utilized. Studies of these mutations have identified amino acid residues that are metal ligands, as well as those residues that are at or near either the quinol oxidase (Q_o) site or the quinol reductase (Q_i) site. The postulate that these two Q-sites are located on opposite sides of the membrane is supported by these studies. Current research is directed at exploring the details of the catalytic mechanism, the nature of the subunit interactions, and the assembly of this enzyme.     

The terminal oxidases of Paracoccus denitrificans

Three distinct types of terminal oxidases participate in the aerobic respiratory pathways of Paracoccus denitrificans. Two alternative genes encoding sub unit I of the aa₃-type cytochrome c oxidase have been isolated before, namely ctaDI and ctaDII. Each of these genes can be expressed separately to complement a double mutant (ActaDI, ActaDII), indicating that they are isoforms of subunit I of the aa₃-type oxidase. The genomic locus of a quinol oxidase has been isolated: cyoABC. Thisprotohaem-containing oxidase, called cytochrome bb₃, is the oniy quinoi oxidase expressed under the conditions used, in a triple oxidase mutant (ActaDI, ActaDII, cyoB::Km^R) an alternative cyto-chrome c oxidase has been characterized; this cbb₃-type oxidase has been partially purified. Both cytochrome aa₃ and cytochrome bb₃ are redox-driven proton pumps. The proton-pumping capacity of cytochrome cbb₃ has been analysed; arguments for and against the active transport of protons by this novel oxidase complex are discussed.     

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.     

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     

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.     

Purification and characterization of arsenite oxidase from Arthrobacter sp.

The chemolithoautotroph, Arthrobacter sp.15b oxidizes arsenite to arsenate using a membrane bound arsenite oxidase. The enzyme arsenite oxidase is purified to its homogeneity and identified using MALDI-TOF MS analysis. Upon further characterization, it was observed that the enzyme is a heterodimer showing native molecular mass as ~100 kDa and appeared as two subunits of ~85 kDa LSU and 14 kDa SSU on SDS–PAGE. The V _max and K _m values of the enzyme was found to be 2.45 μM (AsIII)/min/mg) and 26 μM, respectively. The purified enzyme could withstand wide range of pH and temperature changes. The enzyme, however, gets deactivated in the presence of 1 mM of DEPC suggesting the involvement of histidine at the binding site of the enzyme. The peptide analysis of large sub unit of the enzyme showed close match with the arsenite oxidases of Burkholderia sp. YI019A and arsenite oxidase, Mo-pterin containing subunit of Alcaligenes faecalis. The small subunit, however, differed from other arsenite oxidases and matched only with 2Fe–2S binding protein of Anaplasma phagocytophilum. This indicates that Rieske subunits containing the iron–sulfur clusters present in the large as well as small subunits of the enzyme are integral part of the protein.     

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.     

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.