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Study of locally unfolded forms of cytochrome c by Fourier transform infrared and of H2O2-mediated oxidation of ferricytochrome c and metmyoglobin by mass spectrometry

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Study of locally unfolded forms of cytochrome c by Fourier transform infrared and of H2O2-mediated oxidation of ferricytochrome c and metmyoglobin by mass spectrometry

Filosa, Angelo (2001) Study of locally unfolded forms of cytochrome c by Fourier transform infrared and of H2O2-mediated oxidation of ferricytochrome c and metmyoglobin by mass spectrometry. PhD thesis, Concordia University.

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Abstract

The reaction of hydrogen peroxide (H 2 O 2 ) with metmyoglobin (Mb) and ferricytochrome c (cyt c) forms highly reactive intermediates that are believed to be important in myocardial reperfusion injuries and in triggering apoptosis, respectively. Furthermore, the alkaline isomer(s) of cyt c have been implicated in the apoptotic process. Hence, a detailed study was performed to characterize the locally unfolded forms of cyts c, and to identify reactive intermediates generated in the cyt c and Mb polypeptide upon their reaction with H 2 O 2 . Raising the temperature at neutral pH can induce the alkaline isomerization of cyts c. The intensity of the 695-nm absorption and Fourier transform infrared (FTIR) amide II band as a function of temperature was monitored, revealing that the alkaline form of tuna cyt c appears about 20C̕ lower (V35C̕) than those of horse and cow cyts c (V55-60C̕). The cyts c also exhibit increased susceptibility to tryptic digestion at temperatures approaching their alkaline-transition temperature. Curve-fitting of FTIR amide I ' bands revealed essentially identical secondary structure in horse and cow cyts c, whereas splitting of cc-helical absorption was observed in tuna cyt c, indicating the presence of less stable helical structures. Resolution enhanced 2D IR correlation analysis of the FTIR amide I ' band revealed that an extended-chain structure adjacent to the heme unfolds first in tuna cyt c. By monitoring the Soret absorption of cyt c, it was found that the presence of excess H 2 O 2 caused heme degradation with no evidence for the formation of an oxyferryl heme intermediate in cyt c on the stopped-flow time scale (t 1/2 < 1 ms). However, H 2 O 2 -mediated damage of cyt c was inhibited in the presence of cyanide, confirming that the cyt c/H 2 O 2 reaction is ferric heme-mediated. Spin-trapping was coupled to peptide mass mapping (ST/LC/MS) to locate protein-based radicals formed during the reaction of cyt c and Mb with H 2 O 2 . Tyr74 in horse and cow cyts c and Trp33 in tuna cyt c were inferred as possible radical sites formed during the cyt c/H 2 O 2 reaction, while Tyr103 was confirmed and Lys42 was inferred as possible radical sites formed during the Mb/H 2 O 2 reaction.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Thesis (PhD)
Authors:Filosa, Angelo
Pagination:xxi, 167 leaves : ill. (some col.) ; 29 cm.
Institution:Concordia University
Degree Name:Theses (Ph.D.)
Program:Chemistry and Biochemistry
Date:2001
Thesis Supervisor(s):English, Ann M.
ID Code:1417
Deposited By:Concordia University Libraries
Deposited On:27 Aug 2009 13:19
Last Modified:08 Dec 2010 10:20
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