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

Gilmour, Raymond; Prazeres, Susana; McGinnity, Dermot F.; Goodhew, Celia F.; Moura, José J. G.; Moura, Isabel; Pettigrew, Graham W.

doi:10.1111/j.1432-1033.1995.878_a.x

Cited by 32 publications

(31 citation statements)

References 14 publications

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“…1) into the activated state 3 in which substrate could gain access to the P haem [18,19]. The Pap CCP was shown to have two types of Ca 2+ binding site [20]. Site I was of high affinity (K D 1.2 lM) and was occupied in both the resting and mixed valence states.…”

Section: Haem Function and Enzyme Cyclementioning

confidence: 98%

Structure and mechanism in the bacterial dihaem cytochrome c peroxidases

Pettigrew

Echalier

Pauleta

2006

Journal of Inorganic Biochemistry

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100

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Section: Haem Function and Enzyme Cyclementioning

confidence: 98%

Structure and mechanism in the bacterial dihaem cytochrome c peroxidases

Pettigrew

Echalier

Pauleta

2006

Journal of Inorganic Biochemistry

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100

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“…Dimers of both the small electrontransfer proteins and their enzymic redox partners is a recurring theme in bacterial electron transport. Thus, the CCP redox partner of cytochrome c550 [23] and its Pseudomonad counterpart [22] are dimers. Pseudoazurin of T. pantotropha, a very close relative of Paracoccus LMD 52.44, is dimeric [24] as is its redox partner nitrite reductase [25].…”

Section: Discussionmentioning

confidence: 99%

The surface‐charge asymmetry and dimerisation of cytochrome c550 from Paracoccus denitrificans @MM implications for the interaction with cytochrome c peroxidase

Pettigrew

Gilmour

Goodhew

et al. 1998

European Journal of Biochemistry

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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.Keywords : cytochrome; peroxidase ; electron transfer; electrostatic ; dimerisation.Paracoccus denitrificans cytochrome c550 is the electron donor for Paracoccus cytochrome c peroxidase (CCP). We have used these two redox proteins as a model system, with which to investigate and understand electron transfer between proteins. They represent a useful parallel system to the well-studied yeast cytochrome c and yeast CCP [1,2] in that the cytochrome c donors are structurally similar but the peroxidases are quite distinct.Like the yeast peroxidase, the Paracoccus enzyme requires two electrons to restore the active form after oxidation by hydrogen peroxide. In the yeast case, these are probably delivered singly by two ferrocytochrome c molecules sequentially encountering the peroxidase surface. The Paracoccus enzyme contains two haems rather than one, and in separate domains of the protein [3]. One haem is believed to perform an electrontransferring function and the other, a peroxidatic function. With two domains, each carrying an oxidising centre in the intermedi-

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“…In the more complex case of the Pa. denitrificans enzyme, Gilmour et al [16] proposed that this site (which they called site I) is also present but that there is a second Ca 2+ binding site (called site II) which is mostly empty in the isolated enzyme and the re-occupancy of which is required for the switch to the high-spin state and enzyme activity.…”

Section: Introductionmentioning

confidence: 98%

Ca2+ and the bacterial peroxidases: the cytochrome c peroxidase from Pseudomonas stutzeri

et al. 2003

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The production of cytochrome c peroxidase (CCP) from Pseudomonas ( Ps.) stutzeri (ATCC 11607) was optimized by adjusting the composition of the growth medium and aeration of the culture. The protein was isolated and characterized biochemically and spectroscopically in the oxidized and mixed valence forms. The activity of Ps. stutzeri CCP was studied using two different ferrocytochromes as electron donors: Ps. stutzeri cytochrome c(551) (the physiological electron donor) and horse heart cytochrome c. These electron donors interact differently with Ps. stutzeri CCP, exhibiting different ionic strength dependence. The CCP from Paracoccus ( Pa.) denitrificans was proposed to have two different Ca(2+) binding sites: one usually occupied (site I) and the other either empty or partially occupied in the oxidized enzyme (site II). The Ps. stutzeri enzyme was purified in a form with tightly bound Ca(2+). The affinity for Ca(2+) in the mixed valence enzyme is so high that Ca(2+) returns to it from the EGTA which was added to empty the site in the oxidized enzyme. Molecular mass determination by ultracentrifugation and behavior on gel filtration chromatography have revealed that this CCP is isolated as an active dimer, in contrast to the Pa. denitrificans CCP which requires added Ca(2+) for formation of the dimer and also for activation of the enzyme. This is consistent with the proposal that Ca(2+) in the bacterial peroxidases influences the monomer/dimer equilibrium and the transition to the active form of the enzyme. Additional Ca(2+)does affect both the kinetics of oxidation of horse heart cytochrome c (but not cytochrome c(551)) and higher aggregation states of the enzyme. This suggests the presence of a superficial Ca(2+)binding site of low affinity.

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

Cited by 32 publications

References 14 publications

Structure and mechanism in the bacterial dihaem cytochrome c peroxidases

Structure and mechanism in the bacterial dihaem cytochrome c peroxidases

The surface‐charge asymmetry and dimerisation of cytochrome c550 from Paracoccus denitrificans @MM implications for the interaction with cytochrome c peroxidase

Ca2+ and the bacterial peroxidases: the cytochrome c peroxidase from Pseudomonas stutzeri

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