FORUM ORIGINAL RESEARCH COMMUNICATION Chronic Exposure to Sulfide Causes Accelerated Degradation of Cytochrome c Oxidase in Ethylmalonic Encephalopathy Ivano Di Meo, 1 Gigliola Fagiolari, 2 Alessandro Prelle, 3 Carlo Viscomi, 1 Massimo Zeviani, 1 and Valeria Tiranti 1 Abstract Ethylmalonic encephalopathy (EE) is an autosomal recessive, invariably fatal disorder associated with mutations in ETHE1, a gene encoding a mitochondrial sulfur dioxygenase (SDO). The main consequence of the absence of Ethe1-SDO is the accumulation of sulfide (H 2 S) in critical tissues, including colonic mucosa, liver, muscle, and brain. To make progress in the elucidation of the biochemical mechanisms leading to cytochrome c oxidase (COX) deficiency, we (i) generated tissue-specific conditional Ethe1 knockout mice to clarify the different con- tributions of endogenous and exogenous H 2 S production, and (ii) studied the development of H 2 S-driven COX deficiency in Ethe1 = mouse tissues and human cells. Ethe1 = conditional animals displayed COX deficiency limited to the specific targeted tissue. The accumulation of H 2 S over time causes progressive COX deficiency in animal tissues and human cells, which is associated with reduced amount of COX holoenzyme, and of several COX subunits, including mitochondrially encoded cytochrome c oxidase 1 (MTCO1), MTCO2, COX4, and COX5A. This reduction is not paralleled by consistent downregulation in expression of the corresponding mRNAs. Tissue-specific ablation of Ethe1 causes COX deficiency in targeted organs, suggesting that failure in neutralizing endogenous, tissue-specific production of H 2 S is sufficient to cause the biochemical defect but neither to determine a clinical impact nor to induce the biomarker profile typical of EE. The mechanism by which H 2 S causes COX deficiency consists of rapid heme a inhibition and accelerated long-term degradation of COX subunits. However, the pleiotropic devastating effects of H 2 S accumulation in EE cannot be fully explained by the sole defect of COX in critical tissues, but are likely consequent to several toxic actions on a number of enzymatic activities in different tissues, including endothelial lining of the small vessels, leading to multiorgan failure. Antioxid. Redox Signal. 00, 000–000. Introduction E thylmalonic encephalopathy (EE; OMIM No. 602473) is an autosomal recessive fatal infantile disease caused by accumulation of sulfide, H 2 S, a mitochondrial poison produced exogenously by the anaerobic enterobacterial flora and synthesized endogenously in various mammalian tissues. Failure to detoxify sulfide is due to the absence or malfunctioning of a mitochondrial sulfur dioxygenase (SDO), encoded by the ETHE1 gene, which is mutated in EE (30). EE has been reported in numerous infants of Mediterra- nean or Arab origin who are clinically characterized by (i) progressive encephalopathy; (ii) chronic diarrhea; and (iii) petechial purpura and severe orthostatic acrocyanosis. Bio- chemically, EE presents an unusual combination of severe deficiency of cytochrome c oxidase (COX), the last component (complex IV, cIV) of the mitochondrial respiratory chain (RC), in both muscle and brain, accumulation in blood of C4 and C5 acylcarnitines and urinary excretion of ethylmalonic acid, the dicarboxylic derivative of butyrate (3). We found that the ETHE1 gene codes for an SDO of the mitochondrial matrix, being part of a mitochondrial oxidative pathway for inorganic sulfur that ultimately converts sulfide into harmless com- pounds, such as thiosulfate and sulfate. This pathway is composed of four enzymes. First, a membrane-bound mito- chondrial sulfide quinone reductase is involved in the initial oxidation of H 2 S and the fixation of its sulfur atom to a sulfur acceptor, with the formation of a persulfide (12). Next, the persulfide compound is further oxidized to sulfite by molec- ular oxygen through the action of ETHE1-SDO. The enzyme rhodanese, which displays a sulfur transferase activity, acts in 1 Unit of Molecular Neurogenetics, Pierfranco and Luisa Mariani Center for Research on Children’s Mitochondrial Disorders, Institute of Neurology ‘‘Carlo Besta’’–IRCCS Foundation, Milan, Italy. 2 Division of Neurology, Dino Ferrari Center, Ospedale Maggiore Policlinico, Ca ` Granda-IRCCS Foundation, Milan, Italy. 3 Neuroscience Department, Azienda Ospedaliera Fatebenefratelli e Oftalmico, Milan, Italy. ANTIOXIDANTS & REDOX SIGNALING Volume 00, Number 0, 2011 ª Mary Ann Liebert, Inc. DOI: 10.1089=ars.2010.3520 1