title: Purification and characterization of the complex I from the respiratory chain of Rhodothermus marinus.
abstract: The rotenone sensitive NADH:menaquinone oxidoreductase (NDH-I or complex I) from the thermohalophilic bacterium Rhodothermus marinus has been purified and characterized. Three of its subunits react with antibodies against 78, 51, and 21.3c kDa subunits of Neurospora crassa complex I. The optimum conditions for NADH dehydrogenase activity are 50 degrees C and pH 8.1, and the enzyme presents a KM of 9 microM for NADH. The enzyme also displays NADH:quinone oxidoreductase activity with two menaquinone analogs, 1,4-naphtoquinone (NQ) and 2,3-dimethyl-1,4-naphtoquinone (DMN), being the last one rotenone sensitive, indicating the complex integrity as purified. When incorporated in liposomes, a stimulation of the NADH:DMN oxidoreductase activity is observed by dissipation of the membrane potential, upon addition of CCCP. The purified enzyme contains 13.5 +/- 3.5 iron atoms and approximately 3.7 menaquinone per FMN. At least five iron-sulfur centers are observed by EPR spectroscopy: two [2Fe-2S](2+/1+) and three [4Fe-4S](2+/1+) centers. By fluorescence spectroscopy a still unidentified chromophore was detected in R. marinus complex I.
authors.affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Rua da Quinta Grande 6, 2780-156 Oeiras, Portugal.
authors.forename: Andreia S
authors.initials: AS
authors.lastname: Fernandes
authors.forename: Manuela M
authors.initials: MM
authors.lastname: Pereira
authors.forename: Miguel
authors.initials: M
authors.lastname: Teixeira
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D042967:Electron Transport Complex I; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology*; D007700:Kinetics; D008081:Liposomes; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D021122:Protein Subunits; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
chemical_list: D008081:Liposomes; D021122:Protein Subunits; D009243:NAD; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I