Tsaprailis, George (1997) Probing the redox-active residues in cytochrome c peroxidase. PhD thesis, Concordia University.
The reaction of cytochrome c peroxidase (CCP) with H 2 O 2 results in compound I formation, where the two oxidizing equivalents of H 2 O 2 are stored as an oxyferryl heme and a Trp191 radical. Ferrocytochrome c normally reduces compound I back to the resting enzyme, but in the absence of exogenous donors, CCP can reduce up to 20 equivalents of H 2 O 2 . Compound I of horseradish peroxidase (HRP) does not form a protein radical, and is unlikely to store oxidizing equivalents on its polypeptide. The conformational states in denaturants of recombinant CCP [CCP(MI)], HRP and their CN-ligated forms were investigated to probe the structural basis of peroxidase polypeptide vs heme reactivity. Despite similar structures, the kinetic stabilities and conformational states of CCP(MI) and HRP were found to be significantly different. The role of Trp residues as endogenous electron donors in yeast CCP, CCP(MI), and two active site mutants (W51F and W191F) was examined by protein steady-state fluorescence. Compound I and more highly oxidized forms were formed by adding 2, 6, and 20 equivalents of H 2 O 2 to the proteins in the absence of exogenous donors. Loss of protein fluorescence following protein denaturation in 8 M urea at pH 1.5 was correlated with Trp oxidation. The fluorescence data confirmed Trp191 radical formation in compound I, suggested that Trp5l becomes redox active when >2 equivalents of H 2 O 2 are reduced, and that V3, 4, 2.5 and 2 Trps were lost in CCP, CCP(MI), W51F and W191F, respectively, following addition of 20 equivalents of H 2 O 2 . Activity loss in the H 2 O 2 -oxidized proteins paralleled Trp loss, and correlated with their H 2 O 2 titers. SDS-PAGE revealed 40-75% crosslinking in H 2 O 2 -oxidized W51F, 0-35% in CCP and CCP(MI), and 30-35% in W191F. On-line HPLC-ESI-MS analysis of proteolytic, digests of crosslinked W191F revealed that peptides T 6 (residues 30-48) and T 26 (residues 227-243) formed T 6 --T 6 and T 6 --T 26 crosslinks, suggesting that oxidation of exposed Tyr residues in T 6 (Tyr36, 39, 42) is necessary for crosslinking. H 2 O 2 oxidation of CCP(MI) in the presence of the spin trap, MNP, revealed that spin adducts were formed on peptides T 6 , T 21 (residues 150-155; Tyr153) and T 26 (Tyr229, 236). Tyr236 was identified as the major site of spin adduct formation in CCP(MI) by MS sequencing.
|Divisions:||Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry|
|Item Type:||Thesis (PhD)|
|Pagination:||xx, 219 leaves : ill. ; 29 cm.|
|Degree Name:||Theses (Ph.D.)|
|Thesis Supervisor(s):||English, Ann M.|
|Deposited By:||Concordia University Libraries|
|Deposited On:||27 Aug 2009 17:11|
|Last Modified:||07 Apr 2017 15:13|
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