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Title: Structure-function relationships in haem proteins : a comparative study of ascorbate peroxidase and leghaemoglobin
Author: Jones, Deborah Karen
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 1999
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Ascorbate peroxidase (APX) and leghaemoglobin (Lba) are two highly-specialised haem proteins found in the root nodule of the soybean which maintain homeostasis of oxygen levels. A bacterial expression system was used to generate APX for detailed analyses. Electronic (298K), EPR (7K) and magnetic circular dichroism (MCD) (298K) spectroscopies are consistent with high-spin haem at 298K with small amounts of low-spin species which become more predominant at low temperatures. The reduction potential of the Fe(III)/(II) couple is -159mV, the first reported for an APX. Steady-state kinetics show a sigmoidal dependence upon ascorbate. Transient kinetics (pH7.0, n = 0.10M, 5 °C) gave values of 3.0 0.1 x 10W1 and 3.0 0.1 x 10mV1 for Compound I formation and reduction respectively. A gene encoding soybean Lba was constructed and expressed in E. coli. Electronic, CD, MCD (298K) and EPR (10K) spectroscopies are consistent with native Lba, with a thermal equilibrium of high- and low-spin species at 298K, with increased low-spin at 77K. The reduction potential (vs. NHE) of Lba was +10mV and of nicotinate-bound Lba was -65mV (pH5.4, u= 0.10M, 25 °C), a drop of 100mV relative to the wild-type protein. A regulatory role for nicotinate, which naturally occurs in the root nodules, is proposed. Site-directed mutagenesis was used to probe the functional properties in response to removal of the distal histidine (His61Ala) and incorporation of a leucine residue onto the proximal side (Leu88Asp) of Lba. Their measured reduction potentials (vs. NHE) were -6mV and -14mV for His61Ala and Leu88Asp respectively (pH7.0, i =0.10M, 25 °C). The peroxidase activities of Lba and the variants show that His61 is critical for peroxidase function, while inclusion of Asp88 has less influence. Comparison of the two proteins shows that they are tailored for specific activities, and that single amino acid substitutions are not sufficient to greatly alter their function.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available