Nitrosative stress results in irreversible inhibition of purified mitochondrial complexes I and III without modification of cofactors.
The effects of both nitric oxide (NO) and peroxynitrite on complexes I (NADH dehydrogenase) and III (cytochrome c reductase) isolated from bovine heart have been examined. EPR signals ("g=2.01") previously detected in association with loss of complex I and III activities in cultured cells and isolated mitochondria subjected to nitrosative stress are shown not to arise from these particular enzymes. Neither NO nor peroxynitrite (ONO(2)(-)) reacts to any appreciable extent with the oxidized forms of flavin mononucleotide, iron-sulfur clusters, or heme moieties found in complexes I and III. However, ONO(2)(-) is readily able to abstract electrons from the reduced forms of both complexes I and III, without any apparent modification of the enzyme cofactors. While no attempt was made in the present study to catalog all the possible modifications, it is clear that ONO(2)(-) can react with the protein moieties of the enzymes. For example, when added in excess, ONO(2)(-) derivatizes a select few tyrosine residues in both complexes I and III forming 3-nitrotyrosine as detected by immunoblots. In the case of complex I, we find a minimum of 3 out of the 46 subunits present were modified (49, approximately 18, and approximately 15kDa); whereas in complex III, 4 out of the 13 subunits stained for 3-nitrotyrosine (46, 27, 7, and 6kDa). Significant irreversible inhibition of activity required the addition of >10(2)-fold excesses of ONO(2)(-) to the enzymes. At 10(3)-fold excess of added ONO(2)(-), the activity of complex I was only diminished by approximately 18%, while a 60% loss of activity was observed for complex III.
Pearce
Linda L
LL
Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA. lpearce@eoh.pitt.edu
Kanai
Anthony J
AJ
Epperly
Michael W
MW
Peterson
Jim
J
D000818:Animals; D015415:Biomarkers; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D042967:Electron Transport Complex I / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D014450:Electron Transport Complex III / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D006321:Heart / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007501:Iron / Q000378:metabolism; D008929:Mitochondria, Heart / Q000187:drug effects / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D030421:Peroxynitrous Acid / Q000378:metabolism; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D013455:Sulfur / Q000378:metabolism
D015415:Biomarkers; D021122:Protein Subunits; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D013455:Sulfur; D006861:Hydrogen Peroxide; D007501:Iron; D042967:Electron Transport Complex I; D014450:Electron Transport Complex III