Electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans containing more than one cytochrome † (original) (raw)
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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.
The Structure of an Electron Transfer Complex Containing a Cytochrome c and a Peroxidase
Journal of Biological Chemistry, 1999
Efficient biological electron transfer may require a fluid association of redox partners. Two noncrystallographic methods (a new molecular docking program and 1 H NMR spectroscopy) have been used to study the electron transfer complex formed between the cytochrome c peroxidase (CCP) of Paracoccus denitrificans and cytochromes c. For the natural redox partner, cytochrome c 550 , the results are consistent with a complex in which the heme of a single cytochrome lies above the exposed electron-transferring heme of the peroxidase. In contrast, two molecules of the nonphysiological but kinetically competent horse cytochrome bind between the two hemes of the peroxidase. These dramatically different patterns are consistent with a redox active surface on the peroxidase that may accommodate more than one cytochrome and allow lateral mobility.
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.
Electron transport reactions in a cytochrome c-deficient mutant of Paracoccus denitrificans
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1989
A mutant of Paracoccus denitriflcans which is deficient in c-type cytochromes grows aerobically with generation times similar to those obtained with a wild-type strain. The aa3-type oxidase is functional in the mutant as judged by spectrophotometric assays of cytochrome c oxidation using the membrane particles and cytochrome aa 3 reduction in whole cells. The cytochrome c oxidase (aa 3-tYpe) of the c-less mutant oxidizes soluble cytochrome c at rates equivalent to those obtained with the wild-type. NADH and succinate oxidase activities of the membrane preparations of the mutant and wild-type are also comparable in the absence of detergent treatment. Exogenous soluble cytochrome c can be both reduced by NADH-and succinate-linked systems and oxidized by cytochrome aa 3 present in membranes of the mutant strain. Rapid overall electron transport can occur in the c-less mutant, suggesting that reactions result from collision of diffusing complexes.
The kinetics of the oxidation of cytochrome c by Paracoccus cytochrome c peroxidase
Biochemical Journal, 1994
In work that is complementary to our investigation of the spectroscopic features of the cytochrome c peroxidase from Paracoccus denitrificans [Gilmour, Goodhew, Pettigrew, Prazeres, Moura and Moura (1993) Biochem. J. 294, 745-752], we have studied the kinetics of oxidation of cytochrome c by this enzyme. The enzyme, as isolated, is in the fully oxidized form and is relatively inactive. Reduction of the high-potential haem at pH 6 with ascorbate results in partial activation of the enzyme. Full activation is achieved by addition of 1 mM CaCl2. Enzyme activation is associated with formation of a high-spin state at the oxidized low-potential haem. EGTA treatment of the oxidized enzyme prevents activation after reduction with ascorbate, while treatment with EGTA of the reduced, partially activated, form abolishes the activity. We conclude that the active enzyme is a mixed-valence form with the low-potential haem in a high-spin state that is stabilized by Ca2. Dilution of the enzyme results in a progressive loss of activity, the extent of which depends on the degree of dilution. Most of the activity lost upon dilution can be recovered after reconcentration. The Mr of the enzyme on molecular-exclusion chromatography is concentration-dependent, with a shift to lower values at lower concentrations. Values of Mr obtained are intermediate between those of a monomer (39 565) and a dimer. We propose that the active form of the enzyme is a dimer which dissociates at high dilution to give inactive monomers. From the activity of the enzyme at different dilutions, a KD of 0.8 /tM can be calculated for the monomerdimer equilibrium. The cytochrome c peroxidase oxidizes horse ferrocytochrome c with first-order kinetics, even at high ferrocytochrome c concentrations. The maximal catalytic-centre activity ('turnover number') under the assay conditions used is 62000 min-', with a half-saturating ferrocytochrome c concentration of 3.3 uM. The corresponding values for the Paracoccus cytochrome c-550 (presumed to be the physiological substrate) are 85000 min-' and 13 ,uM. However, in this case, the kinetics deviate from first-order progress curves at all ferrocytochrome c concentrations. Consideration of the periplasmic environment in Paracoccus denitrificans leads us to propose that the enzyme will be present as the fully active dimer supplied with saturating ferrocytochrome c-550.
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...
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.
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.
European Journal of Biochemistry, 1998
The implications of the dimeric state of cytochrome c550 for its binding to Paracoccus cytochrome c peroxidase and its delivery of the two electrons required to restore the active enzyme during catalysis have been investigated. The amino acid sequence of cytochrome c550 of Paracoccus denitrificans strain LMD 52.44 was determined and showed 21 differences from that of strain LMD 22.21. Based on the X-ray structure of the latter, a structure for the cytochrome c550 monomer from strain 52.44 is proposed and a dipole moment of 945 debye was calculated with an orientation close to the exposed haem edge. The behaviour of the cytochrome on molecular-exclusion chromatography is indicative of an ionic strength-dependent monomer (15 kDa)/dimer (30 kDa) equilibrium that can also be detected by 1 H-NMR spectroscopy. The apparent mass of 50 kDa observed at very low ionic strength was consistent with the presence of a strongly asymmetric dimer. This was confirmed by cross-linking studies, which showed that a cross-linked species of mass 30 kDa on SDS behaved with an apparent mass of 50 kDa on molecular-exclusion chromatography. A programme which carried out and evaluated molecular docking of two monomers to give a dimer generated a most probable dimer in which the monomer dipoles lay almost antiparallel to each other. The resultant dipole moment of the dimer is therefore small. Although this finding calls into question the possibility of preorientation of a strongly asymmetrically charged cytochrome as it collides with a redox partner, the stoichiometry of complex formation with cytochrome c peroxidase as studied by 1 H-NMR spectroscopy shows that it is the monomer that binds.