Gilmour, Raymond

2018-05-14T10:13:04Z

2018-05-14T10:13:04Z

1995

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 ≈ -lOOmV). 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 ImM 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 lmM Ca⁺⁺ is added. The same result can be obtained at pH6 by pretreating the enzyme with EGTA prior to ascorbate-reduction. The spin 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. A comparison of the molecular weight of the enzyme under native and denaturing conditions suggests that the enzyme exists in a monomer/ dimer equilibrium. Dilution of the enzyme results in loss of some of the enzyme activity, suggesting that only the dimer is active. Reconcentration of the enzyme recovers the activity lost upon dilution. From the kinetic data, the KD for the monomer/ dimer equilibrium is estimated at 0.6μM The peroxidase can receive electrons both from the acidic Paracoccus cytochrome c-550 and from the basic mitochondrial cytochrome c. Under conditions where the reaction is rapid, the enzyme exhibits first order kinetics with respect to mitochondrial cytochrome c, even at very high substrate concentrations. Deviation from first order is observed at slow rates with mitochondrial cytochrome c and under all conditions tested with Paracoccus cytochrome c550. The apparent maximal turnover of the enzyme is 62000 min⁻¹ with mitochondrial cytochrome c and 85000 min⁻¹ with Paracoccus c-550. The concentrations required for half-maximal activity are 3.25μM mitochondrial cytochrome c and 13μM Paracoccus c-550. Although the mitochondrial and Paracoccus cytochrome donors are very different in overall charge, an examination of their crystal structures show that they both contain a high concentration of positive charge around their front face. The positive front surface in combination with a negatively charged back hemisphere gives the c550 a large dipole moment which has been calculated as 935 Debye (compared to 342 Debye for tuna cytochrome c). This dipole moment may be important for preorientation of the cytochrome prior to its interaction with the peroxidase. Such a preorientation would allow for an increased number of fruitful collisions.

http://hdl.handle.net/1842/29778

The University of Edinburgh

Annexe Thesis Digitisation Project 2018 Block 18

Already catalogued

Electron transfers associated with the cytochrome c peroxidase of Paracoccus denitrificans

The electron transfers associated with the cytochrome c peroxidase of Paracoccus denitrificans

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy