A predicted structure of the cytochrome c oxidase from Burkholderia pseudomallei (original) (raw)
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Biochemistry, 2000
Heme-copper terminal oxidases use the free energy of oxygen reduction to establish a transmembrane proton gradient. While the molecular mechanism of coupling electron transfer to proton pumping is still under debate, recent structure determinations and mutagenesis studies have provided evidence for two pathways for protons within subunit I of this class of enzymes. Here, we probe the D-pathway by mutagenesis of the cytochrome c oxidase of the bacterium Paracoccus denitrificans; amino acid replacements were selected with the rationale of interfering with the hydrophilic lining of the pathway, in particular its assumed chain of water molecules. Proton pumping was assayed in the reconstituted vesicle system by a stopped-flow spectroscopic approach, allowing a reliable assessment of proton translocation efficiency even at low turnover rates. Several mutations at positions above the cytoplasmic pathway entrance (Asn 131, Asn 199) and at the periplasmic exit region (Asp 399) led to complete inhibition of proton pumping; one of these mutants, N131D, exhibited an ideal decoupled phenotype, with a turnover comparable to that of the wild-type enzyme. Since sets of mutations in other positions along the presumed course of the pathway showed normal proton translocation stoichiometries, we conclude that the D-pathway is too wide in most areas above positions 131/199 to be disturbed by single amino acid replacements.
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.
Journal of Biological Chemistry, 1999
The effect of a single site mutation of Arg-54 to methionine in Paracoccus denitrificans cytochrome c oxidase was studied using a combination of optical spectroscopy, electrochemical and rapid kinetics techniques, and time-resolved measurements of electrical membrane potential. The mutation resulted in a blue-shift of the heme a ␣-band by 15 nm and partial occupation of the low-spin heme site by heme O. Additionally, there was a marked decrease in the midpoint potential of the low-spin heme, resulting in slow reduction of this heme species. A stopped-flow investigation of the reaction with ferrocytochrome c yielded a kinetic difference spectrum resembling that of heme a 3. This observation, and the absence of transient absorbance changes at the corresponding wavelength of the low-spin heme, suggests that, in the mutant enzyme, electron transfer from Cu A to the binuclear center may not occur via heme a but that instead direct electron transfer to the high-spin heme is the dominating process. This was supported by charge translocation measurements where ⌬ generation was completely inhibited in the presence of KCN. Our results thus provide an example for how the interplay between protein and cofactors can modulate the functional properties of the enzyme complex. Cytochrome c oxidase (cf. Ref. 1 for a recent review), the terminal enzyme of the respiratory chain of mitochondria and many aerobic bacteria, catalyzes electron transfer from cytochrome c to molecular oxygen thereby reducing the latter to water. The redox reaction is coupled to proton translocation across the inner mitochondrial or bacterial membrane, resulting in an electrochemical proton gradient, ⌬ H ϩ, across the membrane as shown in Reaction 1.
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
Activation and Catalysis of the Di-Heme Cytochrome c Peroxidase from Paracoccus pantotrophus
Structure, 2006
Bacterial cytochrome c peroxidases contain an electron transferring (E) heme domain and a peroxidatic (P) heme domain. All but one of these enzymes are isolated in an inactive oxidized state and require reduction of the E heme by a small redox donor protein in order to activate the P heme. Here we present the structures of the inactive oxidized and active mixed valence enzyme from Paracoccus pantotrophus. Chain flexibility in the former, as expressed by the crystallographic temperature factors, is strikingly distributed in certain loop regions, and these coincide with the regions of conformational change that occur in forming the active mixed valence enzyme. On the basis of these changes, we postulate a series of events that occur to link the trigger of the electron entering the E heme from either pseudoazurin or cytochrome c 550 and the dissociation of a coordinating histidine at the P heme, which allows substrate access.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1996
The ba 3 quinol oxidase from Paracoccus denitrificans has been purified by a new protocol leading to significantly higher yields than previously reported (Richter et al. (1994) J. Biol. Chem. 269, 23079-23086). In an SDS PAG an additional protein band compared with the previous preparation appears, which can be identified as the major form of subunit II. All protein bands can be assigned to genes of the qox operon by N-terminal sequencing, indicating that the oxidase consists of four subunits. In addition to one heme A, one heme B, and one copper atom, the preparation contains two ubiquinone molecules per enzyme. The oxidase is further characterized by electron paramagnetic resonance (EPR), circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopy.
Biochemistry, 1997
The amino acid sequence of the diheme cytochrome c peroxidase from Paracoccus denitrificans has been determined as the result of sequence analysis of peptides generated by chemical and enzymatic cleavages of the apoprotein. The sequence shows 60% similarity to the cytochrome c peroxidase from Pseudomonas aeruginosa, 39% similarity to an open reading frame encoding a putative triheme c-type cytochrome in Escherichia coli, and remote similarity to the MauG proteins from two methylotrophic bacteria. It is proposed, on the basis of the pattern of conserved residues in the sequences, that a change in iron coordination in the N-terminal heme domain may accompany reduction to the active mixed valence state, a change which may be accompanied by conformational adjustments in the highly conserved interface between the N-and C-terminal domains. These conformational adjustments may also lead to the appearance of a second Ca 2+ binding site in the mixed valence enzyme. The exposed edge of the heme in the C-terminal domain is surrounded by several different patterns of charged residues in the Paracoccus and Pseudomonas enzymes, and this is consistent with the interaction of the former with the highly positively charged front face of the donor cytochrome c-550.