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Peroxidatic & spectrophotometric characteristics of a 65-residue heme peptide derivative from cytochrome c

University of British Columbia

1-65H, a 65 amino acid residue long, heme-containing peptide, was produced by incubating horse heart cytochrome c in a solution containing two-hundred fold excess of CNBr. A combination of CO, CN⁻, Imidazole, SCN⁻ and pH spectral titration studies have demonstrated that the heme binding site of 1-65H is more exposed towards the external environment. A consequence of this increased exposure is the 88 to 875-fold amplification of the peroxidase activity in 1-65H over horse heart cytochrome c_. The binding of exogenous ligands to 1-65H inhibited the peroxidase activity thus demonstrating that a necessary condition for peroxidase activity is increased accessibility of the heme moiety to H₂O₂. However the peroxidase activity of Horse Radish Peroxidase (HRP) is approximately three orders of magnitude greater than rates for 1-65H indicating that heme exposure is not sufficient for full peroxidase function. A second consequence of greater heme exposure is the ease in which exogenous ligands bind to the heme group of 1-65H when compared to hhc. The relative increase in exogenous ligand binding has led to a number of interesting findings. Of special note is the discovery that horse heart cytochrome c has a redox-state dependent affinity for CN⁻ but that 1-65H displayed no such property. As a result, it is proposed that the nature of the protein-bound axial ligands play a vital role in determining the redox-state dependency of heme accessibility. Based on exogenous ligand substitution studies on 1-65H, it is also proposed that the type of axial ligands influences the nature of the Soret absorbance band. In particular, electron-withdrawing axial ligands will reduce the intensity of the Soret absorbance peak while electron-donating ligands will increase the intensity of the Soret absorbance peak.

Text

2010-06-20

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http://hdl.handle.net/2429/25860

Biochemistry and Molecular Biology

University of British Columbia

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