Probing the Physiological Roles of Saccharomyces Cerevisiae Cytochrome C Peroxidase Using Biochemical and Proteomics Approaches

Résumé du livre

Yeast cytochrome c peroxidase (CCP) efficiently catalyzes the reduction of H 2 O 2 to H 2 O by ferrocytochrome c and is presumed to be a key component in cellular redox signaling. The CCP W191F variant does not possess ferrocytochrome c -oxidizing activity but does signal oxidative stress. A biophysical study of the recombinant proteins showed that CCP exhibited greater spectral (absorption and near-UV circular dichroism) changes than CCP W191F over 30 min following exposure to 10 molar equivalents of H 2 O 2 . The stable nitroxyl radical, 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO *), inhibited H 2 O 2 -induced protein crosslinking and accelerated the rate of oxyferryl (Fe IV =O) heme reduction. Diethylenetriaminopentaacetic acid (DTPA) inhibited crosslinking of the mutant but not of wild-type CCP. The mass spectral behavior of TEMPO * in aqueous solutions was investigated prior to characterizing its reaction with the H 2 O 2 -oxidized peroxidases. The electrospray ionization mass spectrum showed that TEMPO +, TEMPOH *+ and TEMPOH 2 + ions were formed in the ionization source. TEMPONa *+ ions were dominant in the presence of sodium and fragmented by cleavage of the weak TEMPO * -Na + bond. Quantum chemistry calculations revealed that protonation unlike sodiation destabilized the electronic structure of TEMPO *, which complicated the mass spectra. The function of CCP in vivo was systematically investigated. Cell survival, antioxidant activity and tolerance to heat and oxidants of wild-type, ccp1? (CCP1 -null-mutant) and ccp1?-ccp1 W191F (ccp1? cells transformed with ccp1 W191F) S. cerevisiae strains were compared. The phenotypic differences provide strong evidence that yeast CCP has separate antioxidant and signaling functions in both exponential- and stationary-phase cells. Mitochondria of wild-type and ccp1? strains of S. cerevisiae in the W303-1B genetic background were isolated for proteome analysis. Over 200 protein spots were detected for both strains on 7×10-cm two-dimensional (2D) gels over the pI range 3--10. Since 2DE image analysis was not reliable for proteins with less than three-fold changes in expression, a metabolic labeling approach for isotope-ratio quantitation of proteomes by mass spectrometry (MS) was developed. The W303-1B strain, a leucine auxotroph, was grown on synthetic complete medium containing natural abundance (H 10 -Leu) or perdeuterated Leu (D 10 -Leu), and the cultures were mixed prior to 2DE separation. The D 10 -Leu label provided an internal mass calibrant for accurate quantitation of Leu-containing tryptic peptides by matrix-assisted laser desorption ionization time-of-flight MS. Using this methodology to measure the stress-response indices to H 2 O 2 of six glycolytic enzymes revealed that glycolysis was initially inhibited but restored within about two hours after challenging exponentially growing yeast cells with 0.4 mM H 2 O 2.