Activation of isolated NADH:ubiquinone reductase I (complex I) from Escherichia coli by detergent and phospholipids. Recovery of ubiquinone reductase activity and changes in EPR signals of iron-sulfur clusters.
NADH:ubiquinone oxidoreductase (NDH-1 or complex I) from Escherichia coli was purified using a combination of anion exchange chromatography and centrifugation in sucrose density gradient. The dependence of enzyme activity on detergent and phospholipids was studied. Artificial hexaammineruthenium reductase activity was not affected by dodecyl maltoside (DDM) and asolectin. Ubiquinone reductase activity had a bell-shape dependence on DDM concentration; 7-10-fold activation could be achieved. Treatment with asolectin subsequently yields additional 2-fold activation with a corresponding increase in the apparent V(max) and without significant changes in apparent K(m). Comparative EPR studies of complex I reduced with NADH, "as prepared" and "activated by asolectin" showed an increase in the signals derived mainly from two [4Fe-4S] clusters in the activated enzyme. One of these signals could be simulated with an axial spectrum with g values of g(xyz)= 1.895, 1.904, 2.05, which corresponds to the parameters reported for the N2 cluster. This data indicates conformational rearrangements of catalytic importance in complex I upon binding of phospholipids.
Sinegina
Liliya
L
Helsinki Bioenergetics Group, Institute of Biotechnology, PO Box 65 (Viikinkaari 1) FIN-00014 University of Helsinki, Finland.
Wikström
Mårten
M
Verkhovsky
Michael I
MI
Verkhovskaya
Marina L
ML
D003902:Detergents; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy; D042967:Electron Transport Complex I / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D042963:Electron Transport Complex II / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D005960:Glucosides / Q000737:chemistry*; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000378:metabolism*; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry*; D017975:Ruthenium Compounds / Q000378:metabolism
D003902:Detergents; D029968:Escherichia coli Proteins; D005960:Glucosides; D007506:Iron-Sulfur Proteins; D010743:Phospholipids; D017975:Ruthenium Compounds; D009243:NAD; C031584:hexammineruthenium; C040358:dodecyl maltoside; D042963:Electron Transport Complex II; D042967:Electron Transport Complex I