The branched respiratory chain in mitochondria from the halotolerant yeast Debaryomyces hansenii contains the classical complexes I, II, III and IV plus a cyanide-insensitive, AMP-activated, alternative-oxidase (AOX). Two additional alternative oxidoreductases were found in this organism: an alternative NADH dehydrogenase (NDH2e) and a mitochondrial isoform of glycerol-phosphate dehydrogenase (MitGPDH). These monomeric enzymes lack proton pump activity. They are located on the outer face of the inner mitochondrial membrane. NDH2e oxidizes exogenous NADH in a rotenone-insensitive, flavone-sensitive, process. AOX seems to be constitutive; nonetheless, most electrons are transferred to the cytochromic pathway. Respiratory supercomplexes containing complexes I, III and IV in different stoichiometries were detected. Dimeric complex V was also detected. In-gel activity of NADH dehydrogenase, mass spectrometry, and cytochrome c oxidase and ATPase activities led to determine the composition of the putative supercomplexes. Molecular weights were estimated by comparison with those from the yeast Y. lipolytica and they were IV2, I-IV, III2-IV4, V2, I-III2, I-III2-IV, I-III2-IV2, I-III2-IV3 and I-III2-IV4. Binding of the alternative enzymes to supercomplexes was not detected. This is the first report on the structure and organization of the mitochondrial respiratory chain from D. hansenii.
Keywords: (Mit)GPDH; 2D; ADP; AMP; AOX; Alternative NADH dehydrogenase; Alternative oxidase; BN; COX; CRR; Debaryomyces hansenii; Dig; Glycerol-phosphate dehydrogenase; IMM; LC-MS; LM; MUC; MW; NADH dehydrogenase activity; NDH; NDH2e; PAGE; PG; ROS; Respiratory chain; SDS; Supercomplexes; adenosine diphosphate; adenosine monophosphate; alternative external NADH dehydrogenase; alternative oxidase; blue-native; cyanide-resistant respiration; cytochrome c oxidase; digitonin; glycerol-phosphate dehydrogenase (mitochondrial isoform); inner mitochondrial membrane; laurylmaltoside; liquid chromatography mass spectrometry; mitochondrial unspecific channel; molecular weight; polyacrylamide-gel electrophoresis; propyl-gallate; reactive oxygen species; second dimension; sodium dodecyl sulfate.
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