title: Chemical and NADH-induced, ROS-dependent, cross-linking between subunits of complex I from Escherichia coli and Thermus thermophilus.
abstract: Complex I of respiratory chains transfers electrons from NADH to ubiquinone, coupled to the translocation of protons across the membrane. Two alternative coupling mechanisms are being discussed, redox-driven or conformation-driven. Using "zero-length" cross-linking reagent and isolated hydrophilic domains of complex I from Escherichia coli and Thermus thermophilus, we show that the pattern of cross-links between subunits changes significantly in the presence of NADH. Similar observations were made previously with intact purified E. coli and bovine complex I. This indicates that, upon reduction with NADH, similar conformational changes are likely to occur in the intact enzyme and in the isolated hydrophilic domain (which can be used for crystallographic studies). Within intact E. coli complex I, the cross-link between the hydrophobic subunits NuoA and NuoJ was abolished in the presence of NADH, indicating that conformational changes extend into the membrane domain, possibly as part of a coupling mechanism. Unexpectedly, in the absence of any chemical cross-linker, incubation of complex I with NADH resulted in covalent cross-links between subunits Nqo4 (NuoCD) and Nqo6 (NuoB), as well as between Nqo6 and Nqo9. Their formation depends on the presence of oxygen and so is likely a result of oxidative damage via reactive oxygen species (ROS) induced cross-linking. In addition, ROS- and metal ion-dependent proteolysis of these subunits (as well as Nqo3) is observed. Fe-S cluster N2 is coordinated between subunits Nqo4 and Nqo6 and could be involved in these processes. Our observations suggest that oxidative damage to complex I in vivo may include not only side-chain modifications but also protein cross-linking and degradation.
authors.affiliation: Medical Research Council Dunn Human Nutrition Unit, Wellcome Trust/MRC building, Hills Road, Cambridge CB2 0XY, UK.
authors.forename: John M
authors.initials: JM
authors.lastname: Berrisford
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Thompson
authors.forename: Leonid A
authors.initials: LA
authors.lastname: Sazanov
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D004227:Dithionite / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D042967:Electron Transport Complex I / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D009243:NAD / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism; D011804:Quinolines / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D016963:Thermus thermophilus / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D021122:Protein Subunits; D011804:Quinolines; D017382:Reactive Oxygen Species; D009243:NAD; D004227:Dithionite; C000470:EEDQ; D042967:Electron Transport Complex I; D004229:Dithiothreitol