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

Lopes, Helena; Pettigrew, Graham W.; Moura, Isabel; Moura, José J. G.

doi:10.1007/s007750050277

Cited by 16 publications

(15 citation statements)

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“…Complementing catalytic signals were also observed at the same potential and reveal cooperative electron transfer (18,19). Furthermore, the bacterial P. denitrificans CcP (PdCcP) has been shown to give irreversible, non-turnover electrochemical signals in bulk phase cyclic voltammetry (20). The irreversibility of both the H and L signals was taken to indicate the redox-linked activation process required for the L active site as shown in Fig.…”

Section: Resultsmentioning

confidence: 92%

“…Such a proposal would render the observed k cat a rate-limiting electron transfer constant, and E cat would be due to the H III/II couple. Yet, for NeCcP there is no indication of the electrochemical irreversibly observed for the Paracoccus enzyme which requires reduction of H to become active (20), and E cat is greater than the value of the H III/II potential. Regarding the issue of irreversibility and electrochemical activation, the catalytic waves we observe have highly reversible shape with respect to both the scan rate and the starting potential of the scan.…”

Section: Resultsmentioning

confidence: 97%

“…The irreversibility of both the H and L signals was taken to indicate the redox-linked activation process required for the L active site as shown in Fig. 1; no catalytic activity was observed for PdCcP (20). In contrast to both of these cases, NeCcP films give reversible catalytic waveshapes (Fig.…”

Section: Resultsmentioning

confidence: 99%

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A Distinctive Electrocatalytic Response from the Cytochrome c Peroxidase of Nitrosomonas europaea

Bradley

Chobot

Arciero

et al. 2004

Journal of Biological Chemistry

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Here the cytochrome c peroxidase (CcP) from Nitrosomonas europaea is examined using the technique of catalytic protein film voltammetry. Submonolayers of the bacterial diheme enzyme at a pyrolytic graphite edge electrode give catalytic, reductive signals in the presence of the substrate hydrogen peroxide. The resulting waveshapes indicate that CcP is bound non-covalently in a highly active configuration. The native enzyme has been shown to possess two heme groups of low and high potential (L and H, ؊260 and ؉450 mV versus hydrogen, respectively), and here we find that the catalytic waves of the N. europaea enzyme have a midpoint potential of >500 mV and a shape that corresponds to a 1-electron process. The signals increase in magnitude with hydrogen peroxide concentration, revealing Michaelis-Menten kinetics and K m ‫؍‬ 55 M. The midpoint potentials shift with substrate concentration, indicating the electrochemically active species observed in our data corresponds to a catalytic species. The potentials also shift with respect to pH, and the pH dependence is interpreted in terms of a two pK a model for proton binding. Together the data show that the electrochemistry of the N. europaea cytochrome c peroxidase is unlike other peroxidases studied to date, including other bacterial enzymes. This is discussed in terms of a catalytic model for the N. europaea enzyme and compared with other cytochrome c peroxidases.

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Section: Resultsmentioning

confidence: 92%

Section: Resultsmentioning

confidence: 97%

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A Distinctive Electrocatalytic Response from the Cytochrome c Peroxidase of Nitrosomonas europaea

Bradley

Chobot

Arciero

et al. 2004

Journal of Biological Chemistry

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“…However, it has so far been unclear which heme group acts as the peroxidatic center and which one acts as the electron transferring heme (4, 33, 34). Two plausible mechanisms have been proposed to date (35,36) and are represented in Fig. 4.…”

Section: Mutagenesis and Mechanistic Considerations; Structural Fleximentioning

confidence: 99%

Structural and Mutagenesis Studies on the Cytochrome c Peroxidase from Rhodobacter capsulatus Provide New Insights into Structure-Function Relationships of Bacterial Di-heme Peroxidases

Smet

Savvides

Horen

et al. 2006

Journal of Biological Chemistry

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Cytochrome c peroxidases (CCP) play a key role in cellular detoxification by catalyzing the reduction of hydrogen peroxide to water. The di-heme CCP from Rhodobacter capsulatus is the fastest enzyme (1060 s ؊1 ), when tested with its physiological cytochrome c substrate, among all di-heme CCPs characterized to date and has, therefore, been an attractive target to investigate structure-function relationships for this family of enzymes. Here, we combine for the first time structural studies with site-directed mutagenesis and spectroscopic studies of the mutant enzymes to investigate the roles of amino acid residues that have previously been suggested to be important for activity. The crystal structure of R. capsulatus at 2.7 Å in the fully oxidized state confirms the overall molecular scaffold seen in other di-heme CCPs but further reveals that a segment of about 10 amino acids near the peroxide binding site is disordered in all four molecules in the asymmetric unit of the crystal. Structural and sequence comparisons with other structurally characterized CCPs suggest that flexibility in this part of the molecular scaffold is an inherent molecular property of the R. capsulatus CCP and of CCPs in general and that it correlates with the levels of activity seen in CCPs characterized, thus, far. Mutagenesis studies support the spin switch model and the roles that Met-118, Glu-117, and Trp-97 play in this model. Our results help to clarify a number of aspects of the debate on structure-function relationships in this family of bacterial CCPs and set the stage for future studies.

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“…Few works dealing with the electrochemistry of other peroxidases have been published, with monohaem CCP being the most studied [24,25]. The first reports on bacterial CCPs, from P. pantotrophus and from Nitrosomonas europaea, were recently published [26,27].…”

Section: Introductionmentioning

confidence: 99%

Mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by P. pantotrophus pseudoazurin: kinetics of intermolecular electron transfer

et al. 2007

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This work reports the direct electrochemistry of Paracoccus pantotrophus pseudoazurin and the mediated catalysis of cytochrome c peroxidase from the same organism. The voltammetric behaviour was examined at a gold membrane electrode, and the studies were performed in the presence of calcium to enable the peroxidase activation. A formal reduction potential, E 0 ¢, of 230 ± 5 mV was determined for pseudoazurin at pH 7.0. Its voltammetric signal presented a pH dependence, defined by pK values of 6.5 and 10.5 in the oxidised state and 7.2 in the reduced state, and was constant up to 1 M NaCl. This small copper protein was shown to be competent as an electron donor to cytochrome c peroxidase and the kinetics of intermolecular electron transfer was analysed. A second-order rate constant of 1.4 ± 0.2 · 10 5 M -1 s -1 was determined at 0 M NaCl. This parameter has a maximum at 0.3 M NaCl and is pH-independent between pH 5 and 9.

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

Cited by 16 publications

References 0 publications

A Distinctive Electrocatalytic Response from the Cytochrome c Peroxidase of Nitrosomonas europaea

A Distinctive Electrocatalytic Response from the Cytochrome c Peroxidase of Nitrosomonas europaea

Structural and Mutagenesis Studies on the Cytochrome c Peroxidase from Rhodobacter capsulatus Provide New Insights into Structure-Function Relationships of Bacterial Di-heme Peroxidases

Mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by P. pantotrophus pseudoazurin: kinetics of intermolecular electron transfer

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