The sequence of the cyo operon indicates substantial structural similarities between the cytochrome o ubiquinol oxidase of Escherichia coli and the aa3-type family of cytochrome c oxidases (original) (raw)
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Deletion of the gene for subunit III leads to defective assembly of bacterial cytochrome oxidase
The EMBO Journal, 1989
Communicated by M.Wikstrom COIH is one of the major subunits in the mitochondrial and a bacterial cytochrome c oxidase, cytochrome aa3. It does not contain any of the enzyme's redox-active metal centres and can be removed from the enzyme without major changes in its established functions. We have deleted the COmI gene from Paracoccus denitificans. The mutant still expresses spectroscopically detectable enzyme almost as the wild-type, but its cytochrome c oxidase activity is much lower. From 50 to 80% of cytochrome a is reduced and its absorption maximum is 2-3 nm blueshifted. The EPR signal of ferric cytochrome a is heterogeneous indicating the presence of multiple cytochrome a species. Proteolysis of the membrane-bound oxidase shows new cleavage sites both in COI and COII. DEAE-chromatography of solubilized enzyme yields fractions that contain a COI + COIH complex and in addition haem-binding, free COI as well as free COII. The mutant phenotype can be complemented by introducing the COIH gene back to cells in a plasmid vector. We conclude that cytochrome oxidase assembles inefficiently in the absence of COm and that this subunit may facilitate a late step in the assembly. The different oxidase species in the mutant represent either accumulating intennediates of the assembly pathway or dissociation products of a labile COI + COIl complex and its conformational variants.
Heme-copper terminal oxidase using both cytochrome c and ubiquinol as electron donors
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2012
The cytochrome c oxidase Cox2 has been purified from native membranes of the hyperthermophilic eubacterium Aquifex aeolicus. It is a cytochrome ba 3 oxidase belonging to the family B of the heme-copper containing terminal oxidases. It consists of three subunits, subunit I (CoxA2, 63.9 kDa), subunit II (CoxB2, 16.8 kDa), and an additional subunit IIa of 5.2 kDa. Surprisingly it is able to oxidize both reduced cytochrome c and ubiquinol in a cyanide sensitive manner. Cox2 is part of a respiratory chain supercomplex. This supercomplex contains the fully assembled cytochrome bc 1 complex and Cox2. Although direct ubiquinol oxidation by Cox2 conserves less energy than ubiquinol oxidation by the cytochrome bc 1 complex followed by cytochrome c oxidation by a cytochrome c oxidase, ubiquinol oxidation by Cox2 is of advantage when all ubiquinone would be completely reduced to ubiquinol, e.g., by the sulfide∶quinone oxidoreductase, because the cytochrome bc 1 complex requires the presence of ubiquinone to function according to the Q-cycle mechanism. In the case that all ubiquinone has been reduced to ubiquinol its reoxidation by Cox2 will enable the cytochrome bc 1 complex to resume working. cytochrome c oxidase | quinol oxidase | cyanide inhibition | protein complex interaction
Biochemistry, 1993
The bo-type ubiquinol oxidase of Escherichia coli is a member of the superfamily of hemecopper oxidases which also includes the aa3-type cytochrome c oxidases. The oxygen-binding binuclear center of cytochrome bo is located in subunit I and consists of a heme (heme 0; heme a3 in the aa3-type oxidases) and a copper (CUB). Previous spectroscopic studies have shown that heme o is bound to the protein via a single histidine residue. Site-directed mutagenesis of conserved histidine residues in subunit I has identified two residues (H284 and H419) which are candidates for the ligand of heme 0, while spectroscopic studies of mutants at H284 definitively demonstrated that this residue cannot be the axial ligand. Consequently, the single remaining conserved histidine in subunit I (H419) was assigned as the ligand for the heme of the binuclear center. In this paper, this assignment is tested by characterization of additional mutants in which the putative heme o axial ligand, H419, is replaced by other amino acids. All mutations at H419 result in the loss of enzyme activity. Analyses via UV-visible and Fourier transform infrared spectroscopies reveal that substantial perturbation has occurred at the binuclear center as a result of the amino acid substitutions. In contrast with the wild-type enzyme, the mutant enzymes bind very little carbon monoxide. Three other amino acid residues which are potential ligands for hemeo are shown to be nonessential for enzyme activity. Mutations in these residues do not perturb the UV-visible or FTIR spectroscopic characteristics of the enzyme. These results are consistent with the assignment of H419 as the axial ligand of heme o in the E. coli enzyme and, by analogy, heme a3 in the aa3-type cytochrome c oxidases. The cytochrome bo quinol oxidase from Escherichia coli and the aa3-type cytochrome c oxidases are members of a superfamily of structurally related heme-copper respiratory oxidases (Chepuri et al., 1990b; Saraste et al., 1989). The heme-copper binuclear center, the site of oxygen reduction to water, is diagnostic of this class of respiratory oxidases, and consists of a high-spin heme magnetically coupled to a copper, denoted CUB (Chepuri et al., 1990a; Hill et al., 1992; Hosler et al., 1993; Woodruff, 1993). A second heme, which is sixcoordinate and low-spin, is also present in all members of this superfamily. Vectorial proton translocation across the membrane bilayer appears to be another common feature of the heme-copper oxidases (
Biochemistry, 1993
The bo-type ubiquinol oxidase of Escherichia coli is a member of the superfamily of structurally related heme-copper respiratory oxidases. The members of this family, which also includes the aa3-type cytochrome c oxidases, contain at least two heme prosthetic groups, a six-coordinate low-spin heme, and a high-spin heme. The high-spin heme is magnetically coupled to a copper, CUB, forming a binuclear center which is the site of oxygen reduction to water. Vectorial proton translocation across the membrane bilayer appears to be another common feature of this superfamily of oxidases. It has been proposed previously that the two adjacent histidines in putative transmembrane helix VI1 (H333 and H334 in the E. coli sequence) a Abstract published in Advance ACS Abstracts, October 15, 1993. Abbreviations: CO, carbon monoxide; Cu, copper; C u -m , carbonmonoxy adduct of copper; E,, midpoint potential; EPR, electron paramagnetic resonance; Fe, heme iron; Fe--C=--O, carbonmonoxy adduct of heme; FTIR, Fourier transform infrared; Aullzand BWHH, full width of stretching frequency band at half-maximal height.
Journal of Biological Chemistry
Cyanide-binding to the heme-copper binuclear center of bo-type ubiquinol oxidase from Escherichia coli was investigated with Fourier transform-infrared and EPR spectroscopies. Upon treatment of the air-oxidized CNinhibited enzyme with excess sodium dithionite, a 12 C-14 N stretching vibration at 2146 cm ؊1 characteristic of the Fe O 3؉ -C؍N-Cu B 2؉ bridging structure was quickly replaced with another stretching mode at 2034.5 cm ؊1 derived from the Fe O 2؉ -C؍N moiety. The presence of ubiquinone-8 or ubiquinone-1 caused a gradual autoreduction of the metal center(s) of the air-oxidized CNinhibited enzyme and a concomitant appearance of a strong cyanide stretching band at 2169 cm ؊1 . This 2169 cm ؊1 species could not be retained with a membrane filter (molecular weight cutoff ؍ 10,000) and showed unusual cyanide isotope shifts and a D 2 O shift. These observations together with metal content analyses indicate that the 2169 cm ؊1 band is due to a Cu B ⅐CN complex released from the enzyme. The same species could be produced by anaerobic partial reduction of the CN-inhibited ubiquinol oxidase and, furthermore, of the CNinhibited cytochrome c oxidase; but not at all from the fully reduced CN-inhibited enzymes. These findings suggest that there is a common intermediate structure at the binuclear center of heme-copper respiratory enzymes in the partially reduced state from which the Cu B center can be easily released upon cyanide-binding.