Nitrosative stress affects mitochondrial respiratory chain complex II and complex IV assemblies in Saccharomyces cerevisiae: S-nitrosylation of complex II

Abstract

The elevated level of nitric oxide (NO) and reactive nitrogen species (RNS) induce nitrosative stress in cells and inhibit mitochondrial respiration. Reports showed that RNS rapidly inactivate complex I, followed by inhibition of complex II, III and IV in isolated mitochondria. However, the mechanism(s) by which NO and RNS inhibit these complexes still unclear. In this study facultative anaerobic yeast Saccharomyces cerevisiae has been used for investigating mitochondrial respiratory dysfunction under nitrosative stress, as four out of five mitochondrial oxidative phosphorylation complexes i.e. complexes II, III, IV and V are structurally conserved from yeast to human. Using microbiological growth assays, we showed that S. cerevisiae wild type W3O3 cells treated with graded concentration of sodium nitroprusside (SNP) and S-Nitrosoglutathione (GSNO) induce nitrosative stress, and cell growth was severely compromised under the respiratory proficient rich glycerol-ethanol media. Both the whole cell and the mitochondrial oxygen consumption rates were also significantly compromised under nitrosative stress. Surprisingly, mitochondrial respiratory chain complex II succinate dehydrogenase (SDH) of S. cerevisiae was found S-nitrosylated and therefore inactivated under nitrosative stress. Endogenous RNS produced by S-nitrosoglutathione reductase mutant cells of S. cerevisiae also showed increased S-nitrosylation of SDH. Complex III and IV activities were irreversibly inhibited in S. cerevisiae under nitrosative stress. Interestingly, protein tyrosine nitration was also enhanced in mitochondria in a dose dependent manner upon SNP treatment. Reduced expressions of both Sdh2 (succinate dehydrogenase subunit-2) and Cox2 (mitochondrial complex IV subunit) were observed at the transcription and translation level in S. cerevisiae under nitrosative stress. Blue Native-PAGE followed by Western blotting analysis, further revealed significantly reduced native complex II and the complex III and IV containing super-complexes assemblies in consequences of nitrosative stress in S. cerevisiae. Henceforth, the present in vivo study provides for the first-time novel information on the modification of mitochondrial complexes under nitrosative stress which in turn regulates the mitochondrial respiratory chain complexes assembly in S. cerevisiae.