Sequence analysis of the cbb3 oxidases and an atomic model for the Rhodobacter sphaeroides enzyme

The cbb3-type oxidases are members of the heme-copper oxidase superfamily, distant by sequence comparisons, but sharing common functional characteristics. To understand the minimal common properties of the superfamily, and to learn about cbb3-type oxidases specifically, we have analyzed a wide set of heme-copper oxidase sequences and built a homology model of the catalytic subunit of the cbb3 oxidase from Rhodobacter sphaeroides. We conclude that with regard to the active site surroundings, the cbb3 oxidases greatly resemble the structurally known oxidases, while major differences are found in three segments: the additional N-terminal stretch of ca. 60 amino acids, the segment following helix 3 to the end of helix 5, and the C-terminus from helix 11 onward. The conserved core contains the active site tyrosine and also an analogue of the K-channel of proton transfer, but centered on a well-conserved histidine in the lower part of helix 7. Modeling the variant parts of the enzyme suggests that two periplasmic loops (between helices 3 and 4 and between helices 11 and 12) could interact with each other as a part of the active site structure and might have an important role in proton pumping. An analogue of the D-channel is not found, but an alternative channel might form around helix 9. A preliminary packing model of the trimeric enzyme is also presented.     

Helix switching of a key active-site residue in the cytochrome cbb3 oxidases

In the respiratory chains of mitochondria and many aerobic prokaryotes, heme-copper oxidases are the terminal enzymes that couple the reduction of molecular oxygen to proton pumping, contributing to the protonmotive force. The cbb(3) oxidases belong to the superfamily of enzymes that includes all of the heme-copper oxidases. Sequence analysis indicates that the cbb(3) oxidases are missing an active-site tyrosine residue that is absolutely conserved in all other known heme-copper oxidases. In the other heme-copper oxidases, this tyrosine is known to be subject to an unusual post-translational modification and to play a critical role in the catalytic mechanism. 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. Using homology modeling, quantum chemistry, and molecular dynamics, a model of the structure of subunit I of a cbb(3) oxidase (Vibrio cholerae) was constructed. The model predicts that a tyrosine residue structurally analogous to the active-site tyrosine in other oxidases is present in the cbb(3) oxidases but that the tyrosine originates from a different transmembrane helix within the protein. The predicted active-site tyrosine is conserved in the sequences of all of the known cbb(3) oxidases. Mutagenesis of the tyrosine to phenylalanine in the V. cholerae oxidase resulted in a fully assembled enzyme with nativelike structure but lacking catalytic activity. These findings strongly suggest that all of the heme-copper oxidases utilize the same catalytic mechanism and provide an unusual example in which a critical active-site residue originates from different places within the primary sequence for different members of the same superfamily.     

Site-directed mutagenesis of five conserved residues of subunit i of the cytochrome cbb3 oxidase in Rhodobacter capsulatus

Cytochrome cbb(3) oxidase is a member of the heme-copper oxidase superfamily that catalyses the reduction of molecular oxygen to the water and conserves the liberated energy in the form of a proton gradient. Comparison of the amino acid sequences of subunit I from different classes of heme-copper oxidases showed that transmembrane helix VIII and the loop between transmembrane helices IX and X contain five highly conserved polar residues; Ser333, Ser340, Thr350, Asn390 and Thr394. To determine the relationship between these conserved amino acids and the activity and assembly of the cbb(3) oxidase in Rhodobacter capsulatus, each of these five conserved amino acids was substituted for alanine by site-directed mutagenesis. The effects of these mutations on catalytic activity were determined using a NADI plate assay and by measurements of the rate of oxygen consumption. The consequence of these mutations for the structural integrity of the cbb(3) oxidase was determined by SDS-PAGE analysis of chromatophore membranes followed by TMBZ staining. The results indicate that the Asn390Ala mutation led to a complete loss of enzyme activity and that the Ser333Ala mutation decreased the activity significantly. The remaining mutants cause a partial loss of catalytic activity. All of the mutant enzymes, except Asn390Ala, were apparently correctly assembled and stable in the membrane of the R. capsulatus.     

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.     

Molecular and spectroscopic analysis of the cytochrome cbb(3) oxidase from Pseudomonas stutzeri

Cytochrome cbb(3) oxidase, a member of the heme-copper oxidase superfamily, is characterized by its high affinity for oxygen while retaining the ability to pump protons. These attributes are central to its proposed role in the microaerobic metabolism of proteobacteria. We have completed the first detailed spectroscopic characterization of a cytochrome cbb(3) oxidase, the enzyme purified from Pseudomonas stutzeri. A combination of UV-visible and magnetic CD spectroscopies clearly identified four low-spin hemes and the high-spin heme of the active site. This heme complement is in good agreement with our analysis of the primary sequence of the ccoNOPQ operon and biochemical analysis of the complex. Near-IR magnetic CD spectroscopy revealed the unexpected presence of a low-spin bishistidine-coordinated c-type heme in the complex. This was shown to be one of two c-type hemes in the CcoP subunit by separately expressing the subunit in Escherichia coli. Separate expression of CcoP also allowed us to unambiguously assign each of the signals associated with low-spin ferric hemes present in the X-band EPR spectrum of the oxidized enzyme. This work both underpins future mechanistic studies on this distinctive class of bacterial oxidases and raises questions concerning the role of CcoP in electron delivery to the catalytic subunit.     

Stabilization of the peroxy intermediate in the oxygen splitting reaction of cytochrome cbb(3)

The proton-pumping cbb(3)-type cytochrome c oxidases catalyze cell respiration in many pathogenic bacteria. For reasons not yet understood, the apparent dioxygen (O(2)) affinity in these enzymes is very high relative to other members of the heme-copper oxidase (HCO) superfamily. Based on density functional theory (DFT) calculations on intermediates of the oxygen scission reaction in active-site models of cbb(3)- and aa(3)-type oxidases, we find that a transient peroxy intermediate (I(P), Fe[III]-OOH(-)) is ~6kcal/mol more stable in the former case, resulting in more efficient kinetic trapping of dioxygen and hence in a higher apparent oxygen affinity. The major molecular basis for this stabilization is a glutamate residue, polarizing the proximal histidine ligand of heme b(3) in the active site.     

Gene cluster of Rhodothermus marinus high-potential iron-sulfur Protein: oxygen oxidoreductase, a caa(3)-type oxidase belonging to the superfamily of heme-copper oxidases

The respiratory chain of the thermohalophilic bacterium Rhodothermus marinus contains an oxygen reductase, which uses HiPIP (high potential iron-sulfur protein) as an electron donor. The structural genes encoding the four subunits of this HiPIP:oxygen oxidoreductase were cloned and sequenced. The genes for subunits II, I, III, and IV (named rcoxA to rcoxD) are found in this order and seemed to be organized in an operon of at least five genes with a terminator structure a few nucleotides downstream of rcoxD. Examination of the amino acid sequence of the Rcox subunits shows that the subunits of the R. marinus enzyme have homology to the corresponding subunits of oxidases belonging to the superfamily of heme-copper oxidases. RcoxB has the conserved histidines involved in binding the binuclear center and the low-spin heme. All of the residues proposed to be involved in proton transfer channels are conserved, with the exception of the key glutamate residue of the D-channel (E(278), Paracoccus denitrificans numbering). Analysis of the homology-derived structural model of subunit I shows that the phenol group of a tyrosine (Y) residue and the hydroxyl group of the following serine (S) may functionally substitute the glutamate carboxyl in proton transfer. RcoxA has an additional sequence for heme C binding, after the Cu(A) domain, that is characteristic of caa(3) oxidases belonging to the superfamily. Homology modeling of the structure of this cytochrome domain of subunit II shows no marked electrostatic character, especially around the heme edge region, suggesting that the interaction with a redox partner is not of an electrostatic nature. This observation is analyzed in relation to the electron donor for this caa(3) oxidase, the HiPIP. In conclusion, it is shown that an oxidase, which uses an iron-sulfur protein as an electron donor, is structurally related to the caa(3) class of heme-copper cytochrome c oxidases. The data are discussed in the framework of the evolution of oxidases within the superfamily of heme-copper oxidases.     

The caa(3) terminal oxidase of Rhodothermus marinus lacking the key glutamate of the D-channel is a proton pump

The thermohalophilic bacterium Rhodothermus marinus expresses a caa(3)-type dioxygen reductase as one of its terminal oxidases. The subunit I amino acid sequence shows the presence of all the essential residues of the D- and K-proton channels, defined in most heme-copper oxidases, with the exception of the key glutamate residue located in the middle of the membrane dielectric (E278 in Paracoccus denitrificans). On the basis of homology modeling studies, a tyrosine residue (Y256, R. marinus numbering) has been proposed to act as a functional substitute [Pereira, M. M., Santana, M., Soares, C. M., Mendes, J., Carita, J. N., Fernandes, A. S., Saraste, M., Carrondo, M. A., and Teixeira, M. (1999) Biochim. Biophys. Acta 1413, 1-13]. Here, R. marinus caa(3) oxidase was reconstituted in liposomes and shown to operate as a proton pump, translocating protons from the cytoplasmic side of the bacterial inner membrane to the periplasmatic space with a stoichiometry of 1H(+)/e(-), as in the case in heme-copper oxidases that contain the glutamate residue. Possible mechanisms of proton transfer in the D-channel with the participation of the tyrosine residue are discussed. The observation that the tyrosine residue is conserved in several other members of the heme-copper oxidase superfamily suggests a common alternative mode of action for the D-channel.     

A high-affinity cbb3-type cytochrome oxidase terminates the symbiosis-specific respiratory chain of Bradyrhizobium japonicum.

It has been a long-standing hypothesis that the endosymbiotic rhizobia (bacteroids) cope with a concentration of 10 to 20 nM free O2 in legume root nodules by the use of a specialized respiratory electron transport chain terminating with an oxidase that ought to have a high affinity for O2. Previously, we suggested that the microaerobically and anaerobically induced fixNOQP operon of Bradyrhizobium japonicum might code for such a special oxidase. Here we report the biochemical characteristics of this terminal oxidase after a 27-fold enrichment from membranes of anaerobically grown B. japonicum wild-type cells. The purified oxidase has TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) oxidase activity as well as cytochrome c oxidase activity. N-terminal amino acid sequencing of its major constituent subunits confirmed that presence of the fixN,fixO, and fixP gene products. FixN is a highly hydrophobic, heme B-binding protein. FixO and FixP are membrane-anchored c-type cytochromes (apparent Mrs of 29,000 and 31,000, respectively), as shown by their peroxidase activities in sodium dodecyl sulfate-polyacrylamide gels. All oxidase properties are diagnostic for it to be a member of the cbb3-type subfamily of heme-copper oxidases. The FixP protein was immunologically detectable in membranes isolated from root nodule bacteroids, and 85% of the total cytochrome c oxidase activity in bacteroid membranes was contributed by the cbb3-type oxidase. The Km values for O2 of the purified enzyme and of membranes from different B. japonicum wild-type and mutant strains were determined by a spectrophotometric method with oxygenated soybean leghemoglobin as the sole O2 delivery system. The derived Km value for O2 of the cbb3-type oxidase in membranes was 7 nM, which is six- to eightfold lower than that determined for the aerobic aa3-type cytochrome c oxidase. We conclude that the cbb3-type oxidase supports microaerobic respiration in endosymbiotic bacteroids.     

Spectroscopic and genetic evidence for two heme-Cu-containing oxidases in Rhodobacter sphaeroides.

It has recently become evident that many bacterial respiratory oxidases are members of a superfamily that is related to the eukaryotic cytochrome c oxidase. These oxidases catalyze the reduction of oxygen to water at a heme-copper binuclear center. Fourier transform infrared (FTIR) spectroscopy has been used to examine the heme-copper-containing respiratory oxidases of Rhodobacter sphaeroides Ga. This technique monitors the stretching frequency of CO bound at the oxygen binding site and can be used to characterize the oxidases in situ with membrane preparations. Oxidases that have a heme-copper binuclear center are recognizable by FTIR spectroscopy because the bound CO moves from the heme iron to the nearby copper upon photolysis at low temperature, where it exhibits a diagnostic spectrum. The FTIR spectra indicate that the binuclear center of the R. sphaeroides aa3-type cytochrome c oxidase is remarkably similar to that of the bovine mitochondrial oxidase. Upon deletion of the ctaD gene, encoding subunit I of the aa3-type oxidase, substantial cytochrome c oxidase remains in the membranes of aerobically grown R. sphaeroides. This correlates with a second wild-type R. sphaeroides is grown photosynthetically, the chromatophore membranes lack the aa3-type oxidase but have this second heme-copper oxidase. Subunit I of the heme-copper oxidase superfamily contains the binuclear center. Amino acid sequence alignments show that this subunit is structurally very highly conserved among both eukaryotic and prokaryotic species. The polymerase chain reaction was used to show that the chromosome of R. sphaeroides contains at least one other gene that is a homolog of ctaD, the gene encoding subunit I of the aa3-type cytochrome c oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton translocation by cytochrome c oxidase can take place without the conserved glutamic acid in subunit I

A glutamic acid residue in subunit I of the heme-copper oxidases is highly conserved and has been directly implicated in the O(2) reduction and proton-pumping mechanisms of these respiratory enzymes. Its mutation to residues other than aspartic acid dramatically inhibits activity, and proton translocation is lost. However, this glutamic acid is replaced by a nonacidic residue in some structurally distant members of the heme-copper oxidases, which have a tyrosine residue in the vicinity. Here, using cytochrome c oxidase from Paracoccus denitrificans, we show that replacement of the glutamic acid and a conserved glycine nearby lowers the catalytic activity to <0.1% of the wild-type value. But if, in addition, a phenylalanine that lies close in the structure is changed to tyrosine, the activity rises more than 100-fold and proton translocation is restored. Molecular dynamics simulations suggest that the tyrosine can support a transient array of water molecules that may be essential for proton transfer in the heme-copper oxidases. Surprisingly, the glutamic acid is thus not indispensable, which puts important constraints on the catalytic mechanism of these enzymes.     

Time-resolved single-turnover of caa(3) oxidase from Thermus thermophilus. Fifth electron of the fully reduced enzyme converts O(H) into E(H) state

The oxidative part of the catalytic cycle of the caa(3)-type cytochrome c oxidase from Thermus thermophilus was followed by time-resolved optical spectroscopy. Rate constants, chemical nature and the spectral properties of the catalytic cycle intermediates (Compounds A, P, F) reproduce generally the features typical for the aa(3)-type oxidases with some distinctive peculiarities caused by the presence of an additional 5-th redox-center-a heme center of the covalently bound cytochrome c. Compound A was formed with significantly smaller yield compared to aa(3) oxidases in general and to ba(3) oxidase from the same organism. Two electrons, equilibrated between three input redox-centers: heme a, Cu(A) and heme c are transferred in a single transition to the binuclear center during reduction of the compound F, converting the binuclear center through the highly reactive O(H) state into the final product of the reaction-E(H) (one-electron reduced) state of the catalytic site. In contrast to previous works on the caa(3)-type enzymes, we concluded that the finally produced E(H) state of caa(3) oxidase is characterized by the localization of the fifth electron in the binuclear center, similar to the O(H)→E(H) transition of the aa(3)-type oxidases. So, the fully-reduced caa(3) oxidase is competent in rapid electron transfer from the input redox-centers into the catalytic heme-copper site.     

Glutamate-89 in subunit II of cytochrome bo3 from Escherichia coli is required for the function of the heme-copper oxidase

Recent electrostatics calculations on the cytochrome c oxidase from Paracoccus denitrificans revealed an unexpected coupling between the redox state of the heme-copper center and the state of protonation of a glutamic acid (E78II) that is 25 A away in subunit II of the oxidase. Examination of more than 300 sequences of the homologous subunit in other heme-copper oxidases shows that this residue is virtually totally conserved and is in a cluster of very highly conserved residues at the "negative" end (bacterial cytoplasm or mitochondrial matrix) of the second transmembrane helix. The functional importance of several residues in this cluster (E89II, W93II, T94II, and P96II) was examined by site-directed mutagenesis of the corresponding region of the cytochrome bo(3) quinol oxidase from Escherichia coli (where E89II is the equivalent of residue E78II of the P. denitrificans oxidase). Substitution of E89II with either alanine or glutamine resulted in reducing the rate of turnover to about 43 or 10% of the wild-type value, respectively, whereas E89D has only about 60% of the activity of the control oxidase. The quinol oxidase activity of the W93V mutant is also reduced to about 30% of that of the wild-type oxidase. Spectroscopic studies with the purified E89A and E89Q mutants indicate no perturbation of the heme-copper center. The data suggest that E89II (E. coli numbering) is critical for the function of the heme copper oxidases. The proximity to K362 suggests that this glutamic acid residue may regulate proton entry or transit through the K-channel. This hypothesis is supported by the finding that the degree of oxidation of the low-spin heme b is greater in the steady state using hydrogen peroxide as an oxidant in place of dioxygen for the E89Q mutant. Thus, it appears that the inhibition resulting from the E89II mutation is due to a block in the reduction of the heme-copper binuclear center, expected for K-channel mutants.     

Multi-step assembly pathway of the cbb3-type cytochrome c oxidase complex

The cbb3-type cytochrome c oxidases as members of the heme-copper oxidase superfamily are involved in microaerobic respiration in both pathogenic and non-pathogenic proteobacteria. The biogenesis of these multisubunit enzymes, encoded by the ccoNOQP operon, depends on the ccoGHIS gene products, which are proposed to be specifically required for co-factor insertion and maturation of cbb3-type cytochrome c oxidases. Here, the assembly of the cbb3-type cytochrome c oxidase from the facultative photosynthetic model organism Rhodobacter capsulatus was investigated using blue-native polyacrylamide gel electrophoresis. This process involves the formation of a stable but inactive 210 kDa sub-complex consisting of the subunits CcoNOQ and the assembly proteins CcoH and CcoS. By recruiting monomeric CcoP, this sub-complex is converted into an active 230 kDa CcoNOQP complex. Formation of these complexes and the stability of the monomeric CcoP are impaired drastically upon deletion of ccoGHIS. In a ccoI deletion strain, the 230 kDa complex was absent, although monomeric CcoP was still detectable. In contrast, neither of the complexes nor the monomeric CcoP was found in a ccoH deletion strain. In the absence of CcoS, the 230 kDa complex was assembled. However, it exhibited no enzymatic activity, suggesting that CcoS might be involved in a late step of biogenesis. Based on these data, we propose that CcoN, CcoO and CcoQ assemble first into an inactive 210 kDa sub-complex, which is stabilized via its interactions with CcoH and CcoS. Binding of CcoP, and probably subsequent dissociation of CcoH and CcoS, then generates the active 230 kDa complex. The insertion of the heme cofactors into the c-type cytochromes CcoP and CcoO precedes sub-complex formation, while the cofactor insertion into CcoN could occur either before or after the 210 kDa sub-complex formation during the assembly of the cbb3-type cytochrome c oxidase.     

Comparative genomics and site-directed mutagenesis support the existence of only one input channel for protons in the C-family (cbb3 oxidase) of heme-copper oxygen reductases

Oxygen reductase members of the heme-copper superfamily are terminal respiratory oxidases in mitochondria and many aerobic bacteria and archaea, coupling the reduction of molecular oxygen to water to the translocation of protons across the plasma membrane. The protons required for catalysis and pumping in the oxygen reductases are derived from the cytoplasmic side of the membrane, transferred via proton-conducting channels comprised of hydrogen bond chains containing internal water molecules along with polar amino acid side chains. Recent analyses identified eight oxygen reductase families in the superfamily: the A-, B-, C-, D-, E-, F-, G-, and H-families of oxygen reductases. Two proton input channels, the K-channel and the D-channel, are well established in the A-family of oxygen reductases (exemplified by the mitochondrial cytochrome c oxidases and by the respiratory oxidases from Rhodobacter sphaeroides and Paracoccus denitrificans). Each of these channels can be identified by the pattern of conserved polar amino acid residues within the protein. The C-family (cbb3 oxidases) is the second most abundant oxygen reductase family after the A-family, making up more than 20% of the sequences of the heme-copper superfamily. In this work, sequence analyses and structural modeling have been used to identify likely proton channels in the C-family. The pattern of conserved polar residues supports the presence of only one proton input channel, which is spatially analogous to the K-channel in the A-family. There is no pattern of conserved residues that could form a D-channel analogue or an alternative proton channel. The functional importance of the residues proposed to be part of the K-channel was tested by site-directed mutagenesis using the cbb3 oxidases from R. sphaeroides and Vibrio cholerae. Several of the residues proposed to be part of the putative K-channel had significantly reduced catalytic activity upon mutation: T219V, Y227F/Y228F, N293D, and Y321F. The data strongly suggest that in the C-family only one channel functions for the delivery of both catalytic and pumped protons. In addition, it is also proposed that a pair of acidic residues, which are totally conserved among the C-family, may be part of a proton-conducting exit channel for pumped protons. The residues homologous to these acidic amino acids are highly conserved in the cNOR family of nitric oxide reductases and have previously been implicated as part of a proton-conducting channel delivering protons from the periplasmic side of the membrane to the enzyme active site in the cNOR family. It is possible that the C-family contains a homologous proton-conducting channel that delivers pumped protons in the opposite direction, from the active site to the periplasm.     

The cbb3 oxidases are an ancient innovation of the domain bacteria

A survey of genomes for the presence of gene clusters related to cbb(3) oxidases detected bona fide members of the family in almost all phyla of the domain Bacteria. No archaeal representatives were found. The subunit composition was seen to vary substantially between clades observed on the phylogenetic tree of the catalytic subunit CcoN. The protein diade formed by CcoN and the monoheme cytochrome CcoO appears to constitute the functionally essential "core" of the enzyme conserved in all sampled cbb(3) gene clusters. The topology of the phylogenetic tree contradicts the scenario of a recent origin of cbb(3) oxidases and substantiates the status of this family as a phylogenetic entity on the same level as the other subgroups of the heme-copper superfamily (including nitric oxide reductase). This finding resuscitates and exacerbates the conundrum of the evolutionary origin of heme-copper oxidases.     

Probing the Q-proton pathway of ba3-cytochrome c oxidase by time-resolved Fourier transform infrared spectroscopy

In cytochrome c oxidase, the terminal respiratory enzyme, electron transfers are strongly coupled to proton movements within the enzyme. Two proton pathways (K and D) containing water molecules and hydrophobic amino acids have been identified and suggested to be involved in the proton translocation from the mitochondrial matrix or the bacterial cytoplasm into the active site. In addition to the K and D proton pathways, a third proton pathway (Q) has been identified only in ba3-cytochrome c oxidase from Thermus thermophilus, and consists of residues that are highly conserved in all structurally known heme-copper oxidases. The Q pathway starts from the cytoplasmic side of the membrane and leads through the axial heme a3 ligand His-384 to the propionate of the heme a3 pyrrol ring A, and then via Asn-366 and Asp-372 to the water pool. We have applied FTIR and time-resolved step-scan Fourier transform infrared (TRS2-FTIR) spectroscopies to investigate the protonation/deprotonation events in the Q-proton pathway at ambient temperature. The photolysis of CO from heme a3 and its transient binding to CuB is dynamically linked to structural changes that can be tentatively attributed to ring A propionate of heme a3 (1695/1708 cm(-1)) and to deprotonation of Asp-372 (1726 cm(-1)). The implications of these results with respect to the role of the ring A propionate of heme a3-Asp372-H2O site as a proton carrier to the exit/output proton channel (H2O pool) that is conserved among all structurally known heme-copper oxidases, and is part of the Q-proton pathway in ba3-cytochrome c oxidase, are discussed.     

Effect of mutations of five conserved histidine residues in the catalytic subunit of the cbb3 cytochrome c oxidase on its function

The cbb3 cytochrome c oxidase has the dual function as a terminal oxidase and oxygen sensor in the photosynthetic bacterium, Rhodobacter sphaeroides. The cbb3 oxidase forms a signal transduction pathway together with the PrrBA two-component system that controls photosynthesis gene expression in response to changes in oxygen tension in the environment. Under aerobic conditions the cbb3 oxidase generates an inhibitory signal, which shifts the equilibrium of PrrB kinase/phosphatase activities towards the phosphatase mode. Photosynthesis genes are thereby turned off under aerobic conditions. The catalytic subunit (CcoN) of the R. sphaeroides cbb3 oxidase contains five histidine residues (H214, H233, H303, H320, and H444) that are conserved in all CcoN subunits of the cbb3 oxidase, but not in the catalytic subunits of other members of copper-heme superfamily oxidases. H214A mutation of CcoN affected neither catalytic activity nor sensory (signaling) function of the cbb3 oxidase, whereas H320A mutation led to almost complete loss of both catalytic activity and sensory function of the cbb3 oxidase. H233V and H444A mutations brought about the partial loss of catalytic activity and sensory function of the cbb3 oxidase. Interestingly, the H303A mutant form of the cbb3 oxidase retains the catalytic function as a cytochrome c oxidase as compared to the wild-type oxidase, while it is defective in signaling function as an oxygen sensor. H303 appears to be implicated in either signal sensing or generation of the inhibitory signal to the PrrBA two-component system.     

Complex interactions of carbon monoxide with reduced cytochrome cbb3 oxidase from Pseudomonas stutzeri

Cytochrome cbb(3) oxidase, from Pseudomonas stutzeri, contains a total of five hemes, two of which, a b-type heme in the active site and a hexacoordinate c-type heme, can bind CO in the reduced state. By comparing the cbb(3) oxidase complex and the isolated CcoP subunit, which contains the ligand binding bishistidine-coordinated c-type heme, we have deconvoluted the contribution made by each center to CO binding. A combination of rapid mixing and flash photolysis experiments, coupled with computer simulations, reveals the kinetics of the reaction of c-type heme with CO to be complex as a result of the need to displace an endogenous axial ligand, a property shared with nonsymbiotic plant hemoglobins and some heme-based gas sensing domains. The recombination of CO with heme b(3), unlike all other heme-copper oxidases, including mitochondrial cytochrome c oxidase, is independent of ligand concentration. This observation suggests a very differently organized dinuclear center in which CO exchange between Cu(B) and heme b(3) is significantly enhanced, perhaps reflecting an important determinant of substrate affinity.     

The elusive third subunit IIa of the bacterial B-type oxidases: the enzyme from the hyperthermophile Aquifex aeolicus

The reduction of molecular oxygen to water is catalyzed by complicated membrane-bound metallo-enzymes containing variable numbers of subunits, called cytochrome c oxidases or quinol oxidases. We previously described the cytochrome c oxidase II from the hyperthermophilic bacterium Aquifex aeolicus as a ba(3)-type two-subunit (subunits I and II) enzyme and showed that it is included in a supercomplex involved in the sulfide-oxygen respiration pathway. It belongs to the B-family of the heme-copper oxidases, enzymes that are far less studied than the ones from family A. Here, we describe the presence in this enzyme of an additional transmembrane helix "subunit IIa", which is composed of 41 amino acid residues with a measured molecular mass of 5105 Da. Moreover, we show that subunit II, as expected, is in fact longer than the originally annotated protein (from the genome) and contains a transmembrane domain. Using Aquifex aeolicus genomic sequence analyses, N-terminal sequencing, peptide mass fingerprinting and mass spectrometry analysis on entire subunits, we conclude that the B-type enzyme from this bacterium is a three-subunit complex. It is composed of subunit I (encoded by coxA(2)) of 59000 Da, subunit II (encoded by coxB(2)) of 16700 Da and subunit IIa which contain 12, 1 and 1 transmembrane helices respectively. A structural model indicates that the structural organization of the complex strongly resembles that of the ba(3) cytochrome c oxidase from the bacterium Thermus thermophilus, the IIa helical subunit being structurally the lacking N-terminal transmembrane helix of subunit II present in the A-type oxidases. Analysis of the genomic context of genes encoding oxidases indicates that this third subunit is present in many of the bacterial oxidases from B-family, enzymes that have been described as two-subunit complexes.