Electrochemical study on cytochrome c peroxidase from Paracoccus denitrificans: a shifting pattern of structural and thermodynamic properties as the enzyme is activated

1998 ◽  
Vol 3 (6) ◽  
pp. 632-642 ◽  
Author(s):  
Helder Lopes ◽  
Graham W. Pettigrew ◽  
Isabel Moura ◽  
José J. G. Moura
Keyword(s):  
Cytochrome C ◽  
Thermodynamic Properties ◽  
Paracoccus Denitrificans ◽  
Cytochrome C Peroxidase ◽  
Electrochemical Study

The mechanism of activation of cytochrome c peroxidase from Paracoccus denitrificans

1997 ◽  
Vol 67 (1-4) ◽  
pp. 88
Author(s):  
Isabel Moura ◽  
S. Prazeres ◽  
C. Moreno ◽  
H. Lopes ◽  
G. Pettigrew ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Paracoccus Denitrificans ◽  
Cytochrome C Peroxidase

scholarly journals The cellular location and specificity of bacterial cytochrome c peroxidases

1990 ◽  
Vol 271 (3) ◽  
pp. 707-712 ◽  
Author(s):  
C F Goodhew ◽  
I B Wilson ◽  
D J Hunter ◽  
G W Pettigrew
Keyword(s):  
Cytochrome C ◽  
Paracoccus Denitrificans ◽  
Pseudomonas Stutzeri ◽  
Cytochrome C Peroxidase ◽  
Total Activity ◽  
Gram Negative Bacteria ◽  
Visible Absorption ◽  
Cellular Compartment ◽  
Cytochromes C ◽  
Mitochondrial Cytochromes

The locations of cytochrome c peroxidase and catalase activities in the two Gram-negative bacteria Pseudomonas stutzeri (N.C.I.B. 9721) and Paracoccus denitrificans (N.C.I.B. 8944) were investigated by the production of spheroplasts. In both species the cytochrome c peroxidase was predominantly periplasmic: 92% of total activity in Ps. stutzeri and 98% of nonmembrane-bound activity in Pa. denitrificans were found in this cellular compartment. In contrast, the catalase was mostly in the cytoplasmic fraction. Purification of the Pa. denitrificans cytochrome c peroxidase showed it to be the haem c-containing polypeptide of Mr 42,000 that has already been described by Bosma, Braster, Stouthamer & Van Versefeld [(1987) Eur. J. Biochem. 165, 665-670] but was not identified by them as a peroxidase. The visible-absorption spectra of the enzyme closely resemble those of cytochrome c peroxidase from Pseudomonas aeruginosa but the donor specificity is different, with the Pa. denitrificans enzyme preferring the basic mitochondrial cytochromes c to the acidic cytochromes c-551 and reacting well with the Pa. denitrificans cytochrome c-550.

The Affinity and Specificity of Ca2+-Binding Sites of Cytochrome-c Peroxidase from Paracoccus Denitrificans

1995 ◽  
Vol 234 (3) ◽  
pp. 878-886 ◽  
Author(s):  
Raymond Gilmour ◽  
Susana Prazeres ◽  
Dermot F. Mcginnity ◽  
Celia F. Goodhew ◽  
Jose J. G. Moura ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Binding Sites ◽  
Paracoccus Denitrificans ◽  
Cytochrome C Peroxidase

Spectroscopic studies of cytochrome c peroxidase from Paracoccus denitrificans

1993 ◽  
Vol 51 (1-2) ◽  
pp. 194
Author(s):  
Susana Prazeres ◽  
Natarajan Ravi ◽  
Raymond Gilmour ◽  
Graham Pettigrew ◽  
José J.G. Moura ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Paracoccus Denitrificans ◽  
Cytochrome C Peroxidase ◽  
Spectroscopic Studies

scholarly journals Spectroscopic characterization of cytochrome c peroxidase from Paracoccus denitrificans

1993 ◽  
Vol 294 (3) ◽  
pp. 745-752 ◽  
Author(s):  
R Gilmour ◽  
C F Goodhew ◽  
G W Pettigrew ◽  
S Prazeres ◽  
I Moura ◽  
...  
Keyword(s):  
Cytochrome C ◽  
High Spin ◽  
Spin State ◽  
Time Course ◽  
Paracoccus Denitrificans ◽  
Mixed Valence ◽  
Cytochrome C Peroxidase ◽  
High Spin State ◽  
High Potential ◽  
Intermediate Form

The cytochrome c peroxidase of Paracoccus denitrificans is similar to the well-studied enzyme from Pseudomonas aeruginosa. Like the Pseudomonas enzyme, the Paracoccus peroxidase contains two haem c groups, one high potential and one low potential. The high-potential haem acts as a source of the second electron for H2O2 reduction, and the low-potential haem acts as a peroxidatic centre. Reduction with ascorbate of the high-potential haem of the Paracoccus enzyme results in a switch of the low-potential haem to a high-spin state, as shown by visible and n.m.r. spectroscopy. This high-spin haem of the mixed-valence enzyme is accessible to ligands and binds CN- with a KD of 5 microM. The Paracoccus enzyme is significantly different from that from Pseudomonas in the time course of high-spin formation after reduction of the high-potential haem, and in the requirement for bivalent cations. Reduction with 1 mM ascorbate at pH 6 is complete within 2 min, and this is followed by a slow appearance of the high-spin state with a half-time of 10 min. Thus the process of reduction and spin state change can be easily separated in time and the intermediate form obtained. This separation is also evident in e.p.r. spectra, although the slow change involves an alteration in the low-spin ligation at this temperature rather than a change in spin state. The separation is even more striking at pH 7.5, where no high-spin form is obtained until 1 mM Ca2+ is added to the mixed-valence enzyme. The spin-state switch of the low-potential haem shifts the midpoint redox potential of the high-potential haem by 50 mV, a further indication of haem-haem interaction.

scholarly journals The kinetics of the oxidation of cytochrome c by Paracoccus cytochrome c peroxidase

1994 ◽  
Vol 300 (3) ◽  
pp. 907-914 ◽  
Author(s):  
R Gilmour ◽  
C F Goodhew ◽  
G W Pettigrew ◽  
S Prazeres ◽  
J J Moura ◽  
...  
Keyword(s):  
Cytochrome C ◽  
High Spin ◽  
Spin State ◽  
Paracoccus Denitrificans ◽  
Cytochrome C Peroxidase ◽  
High Spin State ◽  
Enzyme Activation ◽  
First Order ◽  
Ferrocytochrome C ◽  
Kinetics Of

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 M(r) of the enzyme on molecular-exclusion chromatography is concentration-dependent, with a shift to lower values at lower concentrations. Values of M(r) 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 microM 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 62,000 min-1, with a half-saturating ferrocytochrome c concentration of 3.3 microM. The corresponding values for the Paracoccus cytochrome c-550 (presumed to be the physiological substrate) are 85,000 min-1 and 13 microM. 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.

scholarly journals Conversion of the proximal histidine ligand to glutamine restores activity to an inactive mutant of cytochrome c peroxidase.

1992 ◽  
Vol 267 (36) ◽  
pp. 25656-25659
Author(s):  
K Choudhury ◽  
M Sundaramoorthy ◽  
J.M. Mauro ◽  
T.L. Poulos
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Peroxidase
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