Structural Characterization of Paracoccus denitrificans Cytochrome c Peroxidase and Assignment of the Low and High Potential Heme Sites (original) (raw)
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M ssbauer Characterization of Paracoccus denitrificans Cytochrome c Peroxidase
Journal of Biological Chemistry, 1995
Mö ssbauer and electron paramagnetic resonance (EPR) spectroscopies were used to characterize the diheme cytochrome c peroxidase from Paracoccus denitrificans (L.M.D. 52.44). The spectra of the oxidized enzyme show two distinct spectral components characteristic of low spin ferric hemes (S ؍ 1/2), revealing different heme environments for the two heme groups. The Paracoccus peroxidase can be non-physiologically reduced by ascorbate. Mö ssbauer investigation of the ascorbate-reduced peroxidase shows that only one heme (the high potential heme) is reduced and that the reduced heme is diamagnetic (S ؍ 0). The other heme (the low potential heme) remains oxidized, indicating that the enzyme is in a mixed valence, half-reduced state. The EPR spectrum of the half-reduced peroxidase, however, shows two low spin ferric species with g max ؍ 2.89 (species I) and g max ؍ 2.78 (species II). This EPR observation, together with the Mö ssbauer result, suggests that both species are arising from the low potential heme. More interestingly, the spectroscopic properties of these two species are distinct from that of the low potential heme in the oxidized enzyme, providing evidence for heme-heme interaction induced by the reduction of the high potential heme. Addition of calcium ions to the half-reduced enzyme converts species II to species I. Since calcium has been found to promote peroxidase activity, species I may represent the active form of the peroxidatic heme.
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.
Mössbauer Characterization of Paracoccus denitrificans Cytochrome c Peroxidase
Journal of Biological Chemistry, 1995
Mö ssbauer and electron paramagnetic resonance (EPR) spectroscopies were used to characterize the diheme cytochrome c peroxidase from Paracoccus denitrificans (L.M.D. 52.44). The spectra of the oxidized enzyme show two distinct spectral components characteristic of low spin ferric hemes (S ؍ 1/2), revealing different heme environments for the two heme groups. The Paracoccus peroxidase can be non-physiologically reduced by ascorbate. Mö ssbauer investigation of the ascorbate-reduced peroxidase shows that only one heme (the high potential heme) is reduced and that the reduced heme is diamagnetic (S ؍ 0). The other heme (the low potential heme) remains oxidized, indicating that the enzyme is in a mixed valence, half-reduced state. The EPR spectrum of the half-reduced peroxidase, however, shows two low spin ferric species with g max ؍ 2.89 (species I) and g max ؍ 2.78 (species II). This EPR observation, together with the Mö ssbauer result, suggests that both species are arising from the low potential heme. More interestingly, the spectroscopic properties of these two species are distinct from that of the low potential heme in the oxidized enzyme, providing evidence for heme-heme interaction induced by the reduction of the high potential heme. Addition of calcium ions to the half-reduced enzyme converts species II to species I. Since calcium has been found to promote peroxidase activity, species I may represent the active form of the peroxidatic heme.
Proceedings of the National Academy of Sciences, 1997
The aa 3 type cytochrome c oxidase consisting of the core subunits I and II only was isolated from the soil bacterium Paracoccus denitrificans and crystallized as complex with a monoclonal antibody F v fragment. Crystals could be grown in the presence of a number of different nonionic detergents. However, only undecyl-β- d -maltoside and cyclohexyl-hexyl-β- d -maltoside yielded well-ordered crystals suitable for high resolution x-ray crystallographic studies. The crystals belong to space group P2 1 2 1 2 1 and diffract x-rays to at least 2.5 Å (1 Å = 0.1 nm) resolution using synchrotron radiation. The structure was determined to a resolution of 2.7 Å using molecular replacement and refined to a crystallographic R -factor of 20.5% ( R free = 25.9%). The refined model includes subunits I and II and the 2 chains of the F v fragment, 2 heme A molecules, 3 copper atoms, and 1 Mg/Mn atom, a new metal (Ca) binding site, 52 tentatively identified water molecules, and 9 detergent molecules. ...
Mutations in the Ca2+ binding site of the Paracoccus denitrificans cytochrome c oxidase
FEBS Letters, 1999
Recent structure determinations suggested a new binding site for a non-redox active metal ion in subunit I of cytochrome c oxidase both of mitochondrial and of bacterial origin. We analyzed the relevant metal composition of the bovine and the Paracoccus denitrificans enzyme and of bacterial sitedirected mutants in several residues presumably liganding this ion. Unlike the mitochondrial enzyme where a low, substoichiometric content of Ca 2+ was found, the bacterial wild-type (WT) oxidase showed a stoichiometry of one Ca per enzyme monomer. Mutants in Asp-477 (in immediate vicinity of this site) were clearly diminished in their Ca content and the isolated mutant enzyme revealed a spectral shift in the heme a visible absorption upon Ca addition, which was reversed by Na ions. This spectral behavior, largely comparable to that of the mitochondrial enzyme, was not observed for the bacterial WT oxidase. Further structure refinement revealed a tightly bound water molecule as an additional Ca 2+ ligand.
Journal of Biological Chemistry, 1999
Cytochrome c oxidase catalyzes the reduction of oxygen to water. This process is accompanied by the vectorial transport of protons across the mitochondrial or bacterial membrane ("proton pumping"). The mechanism of proton pumping is still a matter of debate. Many proposed mechanisms require structural changes during the reaction cycle of cytochrome c oxidase. Therefore, the structure of the cytochrome c oxidase was determined in the completely oxidized and in the completely reduced states at a temperature of 100 K. No ligand exchanges or other major structural changes upon reduction of the cytochrome c oxidase from Paracoccus denitrificans were observed. The three histidine Cu B ligands are well defined in the oxidized and in the reduced states. These results are hardly compatible with the "histidine cycle" mechanisms formulated previously.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2009
The structure of the two-subunit cytochrome c oxidase from Paracoccus denitrificans has been refined using X-ray cryodata to 2.25 Å resolution in order to gain further insights into its mechanism of action. The refined structural model shows a number of new features including many additional solvent and detergent molecules. The electron density bridging the heme a 3 iron and Cu B of the active site is fitted best by a peroxogroup or a chloride ion. Two waters or OH − groups do not fit, one water (or OH − ) does not provide sufficient electron density. The analysis of crystals of cytochrome c oxidase isolated in the presence of bromide instead of chloride appears to exclude chloride as the bridging ligand. In the D-pathway a hydrogen bonded chain of six water molecules connects Asn131 and Glu278, but the access for protons to this water chain is blocked by Asn113, Asn131 and Asn199. The K-pathway contains two firmly bound water molecules, an additional water chain seems to form its entrance. Above the hemes a cluster of 13 water molecules is observed which potentially form multiple exit pathways for pumped protons. The hydrogen bond pattern excludes that the Cu B ligand His326 is present in the imidazolate form.
Cytochrome-c-binding site on cytochrome oxidase in Paracoccus denitrificans
European journal of biochemistry / FEBS, 1998
To monitor the docking site for cytochrome c on cytochrome oxidase from Paracoccus denitrificans, a series of site-directed mutants in acidic residues exposed on the three largest subunits was constructed, and the purified enzymes were assayed for their steady-state kinetic parameters, their ionic strength dependence, and their fast electron entry kinetics by stopped-flow measurements. Increasing the ionic strength, the maximum of the bell-shaped dependence of the steady-state rate observed for wild type shifts the maximum to lower ionic strength in most of the mutants. The Km determined in steady-state experiments under different conditions is largely increased for most of the subunit II and one of the subunit I mutants, giving evidence that binding is impaired, whereas subunit III residues do not seem to contribute significantly. In addition, the bimolecular rate constant for cytochrome c oxidation under pre-steady state conditions was measured using stopped flow spectroscopy. Tak...
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1998
Cytochrome c oxidase from Paracoccus denitrificans has been purified in two different forms differing in polypeptide composition. An enzyme containing polypeptides I^IV is obtained when the purification procedure is performed in L-Ddodecylmaltoside. If, however, Triton X-100 is used to purify the enzyme under otherwise identical conditions, an enzyme is obtained containing only subunits I^II. The two enzymes are undistinguishable by optical spectroscopy but show significant differences in the transient and steady-state time regimes, as studied by stopped-flow spectroscopy. The observed differences, however, are not due to removal of subunits III and IV, but rather to a specific effect of Triton X-100 which appears to affect cytochrome c binding. From these results it is not expected that subunits III and IV play any significant role in cytochrome c binding and, possibly, in the subsequent electron transfer processes. The results also suggest that both electrostatic and hydrophobic interactions may be important in the initial electron transfer process from cytochrome c.