Abstract

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, ccp1x ( CCP1 -null-mutant) and ccp1x-ccp1 W191F ( ccp1x 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 ccp1x 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{604}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