Identification of a histidine-tyrosine cross-link in the active site of the cbb3-type cytochrome c oxidase from Rhodobacter sphaeroides (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1998
Rhodobacter sphaeroides expresses a bb 3-type quinol oxidase, and two cytochrome c oxidases: cytochrome aa 3 and cytochrome cbb 3. We report here the characterization of the genes encoding this latter oxidase. The ccoNOQP gene cluster of R. sphaeroides contains four open reading frames with high similarity to all ccoNOQP/fixNOQP gene clusters reported so far. CcoN has the six highly conserved histidines proposed to be involved in binding the low spin heme, and the binuclear center metals. ccoO and ccoP code for membrane bound mono-and diheme cytochromes c. ccoQ codes for a small hydrophobic protein of unknown function. Upstream from the cluster there is a conserved Fnr/FixK-like box which may regulate its expression. Analysis of a R. sphaeroides mutant in which the ccoNOQP gene cluster was inactivated confirms that this cluster encodes the cbb 3-type oxidase previously purified. Analysis of proton translocation in several strains shows that cytochrome cbb 3 is a proton pump. We also conclude that cytochromes cbb 3 and aa 3 are the only cytochrome c oxidases in the respiratory chain of R. sphaeroides.
Spectroscopic and genetic evidence for two heme-Cu-containing oxidases in Rhodobacter sphaeroides
Journal of Bacteriology, 1992
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...
FEBS Letters, 1995
A common feature within the heine-copper oxidase superfamily is the dinuclear heine-copper center. Analysis via extended X-ray absorption fine structure (EXAFS) has led to the proposal that sulfur may be bound to CUB, a component of the dinuclear center, and a highly conserved methionine (Mll0 in the E. coil oxidase) in snbanit I has been proposed as the ligand. Recent models of subunit I, however, suggest that this residue is unlikely to be near CUB, but is predicted to be near the low spin heine component of the heine-copper oxidases. In this paper, the role of Mll0 is examined by spectroscopic analyses of sitedirected mutants of the bo~-type oxidase from Escherichia coil The results show that Mll0 is a non-essential residue and suggest that it is probably not near the heine-copper dinuclear center. Key words." ???? subunit I of the heme-copper oxidases. This methionine, which is present in all but three species, has been proposed as a ligand to Cu B in the binuclear center [5]. However, this is incompatible with recent models of subunit I based on site-directed mutagenesis studies of two bacterial heme-copper oxidases, the bo3-type oxidase from E. coli and aa3-type oxidase from Rhodobacter sphaeroides [4,7]. In these models, MII0 is located four residues below the histidine (H106) identified as a ligand for the low spin heine b component of the E. coli oxidase [8,9], which corresponds to the heme a component of the aa3-type oxidases [4,7,10,11]. In this work, the role of Mll0 in the bo3-type oxidase from E. coli was examined by site-directed mutagenesis. The results clearly show that Mll0 is not an essential residue and is unlikely to be near the dinuclear center, consistent with the current models of subunit I of the hemecopper oxidases [4,7,10].
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2012
3-type cytochrome c oxidase aa 3-type cytochrome c oxidase Copper chaperone Copper center assembly Cu A Sco protein The α proteobacter Rhodobacter sphaeroides accumulates two cytochrome c oxidases (CcO) in its cytoplasmic membrane during aerobic growth: a mitochondrial-like aa 3-type CcO containing a di-copper Cu A center and mono-copper Cu B , plus a cbb 3-type CcO that contains Cu B but lacks Cu A. Three copper chaperones are located in the periplasm of R. sphaeroides, PCu A C, PrrC (Sco) and Cox11. Cox11 is required to assemble Cu B of the aa 3type but not the cbb 3-type CcO. PrrC is homologous to mitochondrial Sco1; Sco proteins are implicated in Cu A assembly in mitochondria and bacteria, and with Cu B assembly of the cbb 3-type CcO. PCu A C is present in many bacteria, but not mitochondria. PCu A C of Thermus thermophilus metallates a Cu A center in vitro, but its in vivo function has not been explored. Here, the extent of copper center assembly in the aa 3-and cbb 3type CcOs of R. sphaeroides has been examined in strains lacking PCu A C, PrrC, or both. The absence of either chaperone strongly lowers the accumulation of both CcOs in the cells grown in low concentrations of Cu 2 +. The absence of PrrC has a greater effect than the absence of PCu A C and PCu A C appears to function upstream of PrrC. Analysis of purified aa 3-type CcO shows that PrrC has a greater effect on the assembly of its Cu A than does PCu A C, and both chaperones have a lesser but significant effect on the assembly of its Cu B even though Cox11 is present. Scenarios for the cellular roles of PCu A C and PrrC are considered. The results are most consistent with a role for PrrC in the capture and delivery of copper to Cu A of the aa 3-type CcO and to Cu B of the cbb 3-type CcO, while the predominant role of PCu A C may be to capture and deliver copper to PrrC and Cox11. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.
Proceedings of the National Academy of Sciences, 2005
Sco1 and Cox17 are accessory proteins required for the correct assembly of eukaryotic cytochrome c oxidase. At variance with Sco1, Cox17 orthologs are found only in eukaryotes. We browsed bacterial genomes to search proteins functionally equivalent to Cox17, and we identified a class of proteins of unknown function displaying a conserved gene neighborhood to bacterial Sco1 genes, all sharing a potential metal binding motif H(M)X 10 MX 21 HXM. Two members of this group, DR1885 from Deinococcus radiodurans and CC3502 from Caulobacter crescentus , were expressed, and their interaction with copper was investigated. The solution structure and extended x-ray absorption fine structure data on the former protein reveal that the protein binds copper(I) through a histidine and three Mets in a cupredoxin-like fold. The surface location of the copper-binding site as well as the type of coordination are well poised for metal transfer chemistry, suggesting that DR1885 might transfer copper, takin...
A common coupling mechanism for A-type heme-copper oxidases from bacteria to mitochondria
Proceedings of the National Academy of Sciences
Mitochondria metabolize almost all the oxygen that we consume, reducing it to water by cytochrome c oxidase (CcO). CcO maximizes energy capture into the protonmotive force by pumping protons across the mitochondrial inner membrane. Forty years after the H+/e− stoichiometry was established, a consensus has yet to be reached on the route taken by pumped protons to traverse CcO’s hydrophobic core and on whether bacterial and mitochondrial CcOs operate via the same coupling mechanism. To resolve this, we exploited the unique amenability to mitochondrial DNA mutagenesis of the yeast Saccharomyces cerevisiae to introduce single point mutations in the hydrophilic pathways of CcO to test function. From adenosine diphosphate to oxygen ratio measurements on preparations of intact mitochondria, we definitely established that the D-channel, and not the H-channel, is the proton pump of the yeast mitochondrial enzyme, supporting an identical coupling mechanism in all forms of the enzyme.
pH-Dependent Structural Changes at the Heme-Copper Binuclear Center of Cytochrome c Oxidase
Biophysical Journal, 2001
The resonance Raman spectra of the aa 3 cytochrome c oxidase from Rhodobacter sphaeroides reveal pH-dependent structural changes in the binuclear site at room temperature. The binuclear site, which is the catalytic center of the enzyme, possesses two conformations at neutral pH, assessed from their distinctly different Fe-CO stretching modes in the resonance Raman spectra of the CO complex of the fully reduced enzyme. The two conformations (␣ and ) interconvert reversibly in the pH 6 -9 range with a pKa of 7.4, consistent with Fourier transform infrared spectroscopy measurements done at cryogenic temperatures (D. M. Mitchell, J. P. Shapleigh, A. M. Archer, J. O. Alben, and R. B. Gennis, 1996, Biochemistry 35:9446 -9450). It is postulated that the different structures result from a change in the position of the Cu B atom with respect to the CO due to the presence of one or more ionizable groups in the vicinity of the binuclear center. The conserved tyrosine residue (Tyr-288 in R. sphaeroides, Tyr-244 in the bovine enzyme) that is adjacent to the oxygen-binding pocket or one of the histidines that coordinate Cu B are possible candidates. The existence of an equilibrium between the two conformers at physiological pH and room temperature suggests that the conformers may be functionally involved in enzymatic activity.
Biochemistry, 2006
In the respiratory chains of aerobic organisms, oxygen reductase members of the heme-copper superfamily couple the reduction of O 2 to proton pumping, generating an electrochemical gradient. There are three distinct families of heme-copper oxygen reductases: A-, B-and C-type. The A-and B-type oxygen reductases have an active-site tyrosine that forms a unique crosslinked histidinetyrosine cofactor. In the C-type oxygen reductases (also called cbb 3 oxidases) an analogous activesite tyrosine has recently been predicted by molecular modeling to be located within a different transmembrane helix in comparison to the A-and B-type oxygen reductases. In this work mass spectrometry is used to show that the predicted tyrosine forms a histidine-tyrosine crosslinked cofactor in the active site of the C-type oxygen reductases. This is the first known example of the evolutionary migration of a post-translationally modified active-site residue. It also verifies the presence of a unique cofactor in all three families of proton pumping respiratory oxidases, demonstrating that these enzymes likely share a common reaction mechanism and that the histidinetyrosine cofactor may be a required component for proton pumping.
Biochemistry, 2010
The copper amine oxidases carry out two copper-dependent processes: production of their own redox-active cofactor (2,4,5-trihydroxyphenylalanine quinone, TPQ), and the subsequent oxidative deamination of substrate amines. Because the same active-site pocket must facilitate both reactions, individual active-site residues may serve multiple roles. We have examined the roles of a strictly-conserved active-site tyrosine Y305 in the copper amine oxidase from Hansenula polymorpha kinetically, spetroscopically, and, in the present work, structurally. While the Y305A enzyme is almost identical to the wild-type, a novel, highly oxygenated species replaces TPQ in the Y305F active sites. This new structure not only provides the first direct detection of peroxyintermediates in cofactor biogenesis, but also indicates the critical control of oxidation chemistry that can be conferred by a single active-site residue.