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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.

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.

Cytochrome cbb(3) oxidases are found almost exclusively in Proteobacteria, and represent a distinctive class of proton-pumping respiratory heme-copper oxidases (HCO) that lack many of the key structural features that contribute to the reaction cycle of the intensely studied mitochondrial cytochrome c oxidase (CcO). Expression of cytochrome cbb(3) oxidase allows human pathogens to colonise anoxic tissues and agronomically important diazotrophs to sustain N(2) fixation. We review recent progress in the biochemical characterisation of these distinctive oxidases that lays the foundation for understanding the basis of their proposed high affinity for oxygen, an apparent degeneracy in their electron input pathways and whether or not they acquired the ability to pump protons independently of other HCOs.

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.

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.

Cytochrome cbb(3) oxidase is a member of the haem-copper oxidase superfamily. It is characterized by its high oxygen affinity, while retaining the ability to pump protons. These attributes are central to its proposed role in bacterial microaerobic metabolism. Recent spectroscopic characterization of both the cytochrome cbb(3) oxidase complex from Pseudomonas stutzeri and the dihaem ccoP subunit expressed separately in Escherichia coli has revealed the presence of a low-spin His/His co-ordinated c-type cytochrome. The low midpoint reduction potential of this haem (E(m)<+100 mV), together with its unexpected ability to bind CO in the reduced state at the expense of the distal histidine ligand, raises questions about the role of the ccoP subunit in the delivery of electrons to the active site.

The O2 reaction and the reverse electron transfer of the thermophilic caa3 terminal oxidase of Bacillus stearothermophilus have been studied by laser flash-photolysis. The results show that both reactions, although studied at a temperature of 20 degreesC, far from the optimal temperature of > 60 degreesC for caa3, follow a kinetic behavior essentially identical to that observed with the electrostatic complex between mammalian cyt c and cyt c oxidase. In the O2 reaction cyt a and cyt a3 are very quickly oxidized; cyt a is then re-reduced via CuA, whereas cyt c oxidation is apparently rate-limited by the oxidation of CuA. Upon photodissociation of the mixed valence-CO caa3, reverse electron transfer from the binuclear center to cyt a3+ (tau1 = 3 micros) and CuA2+ (tau2 = 64 micros) is observed, while cyt c is not reduced by any detectable level. These results seem to rule out accounting for enzymatic thermophilicity by altered kinetics of intramolecular electron transfer involving the cyt center in the reduced configuration, which is very fast. On the basis of these results and previous data, we propose that thermophilicity involves an increased activation barrier for the reduction of cyt a3-CuB in the configuration typical of the oxidized site.

Subunit II of cytochrome c oxidase has a C-terminal domain that is exposed to aqueous solution on membrane surface and contains a copper center called CuA. The central part of the cytochrome c binding site is thought to reside in this domain. We have expressed the subunit II fragment of the Paracoccus denitrificans cytochrome c oxidase in a soluble form and studied its interaction with cytochrome c by stopped-flow spectroscopy. The oxidation of cytochrome c by the CuA domain follows monophasic kinetics, indicating the presence of a single kinetically competent binding site. In low ionic strength medium, the domain oxidizes Paracoccus cytochrome c-550 and horse mitochondrial cytochrome c at the rates of 1.5 x 10(6) and 3 x 10(5) M-1 s-1, respectively. The reaction rates are strongly dependent on ionic strength, which must reflect electrostatic interactions within the complex. The KD for the complex between the bacterial cytochrome c and the domain is 1.6 microM; i.e., it is similar to that between the mitochondrial cytochrome c and the intact oxidase, suggesting that both contain the same catalytically competent binding site. Using site-directed mutagenesis, we have identified five conserved residues of the CuA domain that are involved in the cytochrome c binding. Mutations of glutamine 148, glutamate 154, aspartate 206, aspartate 221, or glutamate 246 lead to a 35-85% decrease in the rate of cytochrome c oxidation. The simultaneous substitution of three invariant carboxylic acids (aspartate 206, aspartate 221, and glutamate 246) leads to a 95% decrease in the reaction rate. Conversely, the reaction can be enhanced by removing a positive charge (lysine 219) from the CuA domain.