Direct evidence for S-nitrosation of mitochondrial complex I.
NO* (nitric oxide) is a pleiotropic signalling molecule, with many of its effects on cell function being elicited at the level of the mitochondrion. In addition to the well-characterized binding of NO* to the Cu(B)/haem-a3 site in mitochondrial complex IV, it has been proposed by several laboratories that complex I can be inhibited by S-nitrosation of a cysteine. However, direct molecular evidence for this is lacking. In this investigation we have combined separation techniques for complex I (blue-native gel electrophoresis, Superose 6 column chromatography) with sensitive detection methods for S-nitrosothiols (chemiluminescence, biotin-switch assay), to show that the 75 kDa subunit of complex I is S-nitrosated in mitochondria treated with S-nitrosoglutathione (10 microM-1 mM). The stoichiometry of S-nitrosation was 7:1 (i.e. 7 mol of S-nitrosothiols per mol of complex I) and this resulted in significant inhibition of the complex. Furthermore, S-nitrosothiols were detected in mitochondria isolated from hearts subjected to ischaemic preconditioning. The implications of these results for the physiological regulation of respiration, for reactive oxygen species generation and for a potential role of S-nitrosation in cardioprotection are discussed.
Burwell
Lindsay S
LS
Department of Anesthesiology, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14620, USA.
Nadtochiy
Sergiy M
SM
Tompkins
Andrew J
AJ
Young
Sara
S
Brookes
Paul S
PS
D000818:Animals; D042967:Electron Transport Complex I / Q000378:metabolism*; D019157:Ischemic Preconditioning, Myocardial; D008297:Male; D008928:Mitochondria / Q000737:chemistry* / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism*; D015538:Nitrosation; D051381:Rats; D017207:Rats, Sprague-Dawley; D026403:S-Nitrosothiols / Q000378:metabolism; D012680:Sensitivity and Specificity
D009566:Nitrates; D026403:S-Nitrosothiols; D042967:Electron Transport Complex I