Oxidatively damaged proteins of heart mitochondrial electron transport complexes

Abstract

Protein modifications, such as carbonylation, nitration and formation of lipid peroxidation adducts, e.g. 4-hydroxynonenal (HNE), are products of oxidative damage attributed to reactive oxygen species (ROS). The mitochondrial respiratory chain Complexes I and III have been shown to be a major source of ROS in vitro. Additionally, modifications of the respiratory chain Complexes (I-V) by nitration, carbonylation and HNE adduct decrease their enzymatic activity in vitro. However, modification of these respiratory chain complex proteins due to in vivo basal level ROS generation has not been investigated. In this study, we show a basal level of oxidative damage to specific proteins of adult bovine heart submitochondrial particle (SMP) complexes, and find that most of these proteins are localized in the mitochondrial matrix. We postulate that electron leakage from respiratory chain complexes and subsequent ROS formation may cause damage to specific complex subunits and contribute to long-term accumulation of mitochondrial dysfunction.

Age-related alterations in oxidatively damaged proteins of mouse heart mitochondrial electron transport chain complexes

“…The reaction was initiated with the addition of 40 μM oxaloacetate and the enzyme activity was recorded at 412 nm (ε = 13.6 mM −1 cm −1 ). Rotenone-sensitive Complex I (CI) activity, Malonate-sensitive Complex II (CII) activity, Antimycin A-sensitive Complex III (CIII) activity, KCN-sensitive Complex IV (CIV) activity, and Oligomycin-sensitive Complex V (CV) activities were assayed as described [9,26,33]. Briefly, CI activity was measured at 340 nm (ε = 6.81 mM −1 cm −1 ) in 1 mL reaction mixture containing reaction buffer (50 mM Potassium Phosphate, pH 7.4, 5 mM MgCl 2 and 1 mg/mL BSA), 7-8 μg of sonicated mitochondria, 2 mM KCN, 3.7 μM Antimycin A and 100 μM Q 1 .…”

“…BN-PAGE and SDS-PAGE were carried out by established methods with minor modifications [9,26,38]. Briefly, a 5 to 12% acrylamide gradient was used for the first dimension BN-PAGE, imidazole instead of Bis-Tris was used as a buffer, and Criterion 10-20% 2D-well gels (BioRad, CA) were used for the second dimension SDS-PAGE.…”

“…The proteins were eluted from the gel and digested with trypsin (Promega, WI); the tryptic peptides were then analyzed by MALDI-TOF-TOF [26]. Mass spectral peak data were submitted to the ProFound (Rockefeller University) online search engine for protein identification using the NCBI database.…”

“…Mitochondrial generated ROS, produced by in vivo electron leakage from ETC CI and CIII play a key role in the modification of mitochondrial proteins [18,[21][22][23][24][25]. These modifications have served as molecular markers of oxidative stress [26,27]. In these experiments we identify the oxidatively modified ETC CI-CV proteins in aged mouse heart, whether these proteins accumulate with age and affect ETC complex function.…”

“…Protein modifications caused by ROS include the formation of lipid peroxidation adducts (4-hydroxynonenal or HNE and malondialdehyde or MDA), carbonylation of lysine, arginine, proline, and threonine, and nitration of tyrosine [28][29][30][31]. Oxidatively damaged proteins have been detected and identified by mass spectrometry [9,26,32]. We propose to determine whether such oxidative modifications cause mitochondrial dysfunction associated with aging and age-associated diseases [9,30,[31][32][33].…”