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Title: The electron transfers associated with the cytochrome c peroxidase of Paracoccus denitrificans
Author: Gilmour, R.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1993
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Abstract:
A study of the electron transfers associated with the cytochrome c peroxidase of Paracoccus denitrificans has been made. The peroxidase is similar to the well-studied enzyme from Pseudomonas aeruginosa, although significant differences do exist. Like the Pseudomonas enzyme, the Paracoccus peroxidase contains two haem c groups, one high potential (Em=+226mV) and one low potential (Em≈-100mV). The high potential haem acts as a source of the second electron for hydrogen peroxide reduction and the low potential haem acts as a peroxidatic centre. The fully oxidised form of the Paracoccus enzyme is inactive and does not bind added ligands. Reduction of the high potential haem (by ascorbate treatment) 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 5μM. 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 divalent cations. Reduction with 1mM 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. This separation is also evident in e.p.r. spectra although the slow change involves an alteration in the low spin ligation at this low temperature rather than a change in spin state. The appearance of the high spin state after ascorbate-reduction is correlated with an increase in enzyme activity, suggesting that the mixed-valence, high-spin state of the enzyme is the active form. At pH 7.5 the separation between ascorbate-reduction and spin-state change in the low potential haem is even more striking, no high spin form being obtained until 1 mM Ca++ is added to the mixed valence enzyme. This spectroscopic observation is also reflected in the kinetics where no enzyme activity is seen until 1mM Ca++ is added. The same result can be obtained at pH6 by pretreating the enzyme with EGTA prior to ascorbate-reduction. The state switch of the low potential haem shifts the midpoint redox potential of the high potential haem by 50mV, a further indication of haem-haem interaction.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.651506  DOI: Not available
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