Purification and characterization of the complex I from the respiratory chain of Rhodothermus marinus
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 con...
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| Veröffentlicht in: | Journal of bioenergetics and biomembranes 2002-12, Vol.34 (6), p.413-421 |
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| creator | Fernandes, Andreia S Pereira, Manuela M Teixeira, Miguel |
| description | 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. |
| doi_str_mv | 10.1023/A:1022509907553 |
| format | Article |
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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.</description><identifier>ISSN: 0145-479X</identifier><identifier>EISSN: 1573-6881</identifier><identifier>DOI: 10.1023/A:1022509907553</identifier><identifier>PMID: 12678433</identifier><language>eng</language><publisher>United States: Springer Nature B.V</publisher><subject>Bacteria ; Electron Spin Resonance Spectroscopy ; Electron Transport Complex I ; Enzymes ; Gram-Negative Aerobic Bacteria - enzymology ; Kinetics ; Liposomes ; Molecular Weight ; NAD - metabolism ; NADH, NADPH Oxidoreductases - chemistry ; NADH, NADPH Oxidoreductases - isolation & purification ; NADH, NADPH Oxidoreductases - metabolism ; Protein Subunits ; Rotenone ; Spectrometry, Fluorescence ; Spectrophotometry ; Spectroscopy ; Sulfur</subject><ispartof>Journal of bioenergetics and biomembranes, 2002-12, Vol.34 (6), p.413-421</ispartof><rights>Plenum Publishing Corporation 2002</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c280t-669beac3a2a2e3cfb111f1e7a67a15386eefa9c8ee63c94d378ad7e12e1719fb3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27901,27902</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/12678433$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Fernandes, Andreia S</creatorcontrib><creatorcontrib>Pereira, Manuela M</creatorcontrib><creatorcontrib>Teixeira, Miguel</creatorcontrib><title>Purification and characterization of the complex I from the respiratory chain of Rhodothermus marinus</title><title>Journal of bioenergetics and biomembranes</title><addtitle>J Bioenerg Biomembr</addtitle><description>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.</description><subject>Bacteria</subject><subject>Electron Spin Resonance Spectroscopy</subject><subject>Electron Transport Complex I</subject><subject>Enzymes</subject><subject>Gram-Negative Aerobic Bacteria - enzymology</subject><subject>Kinetics</subject><subject>Liposomes</subject><subject>Molecular Weight</subject><subject>NAD - metabolism</subject><subject>NADH, NADPH Oxidoreductases - chemistry</subject><subject>NADH, NADPH Oxidoreductases - isolation & purification</subject><subject>NADH, NADPH Oxidoreductases - metabolism</subject><subject>Protein Subunits</subject><subject>Rotenone</subject><subject>Spectrometry, Fluorescence</subject><subject>Spectrophotometry</subject><subject>Spectroscopy</subject><subject>Sulfur</subject><issn>0145-479X</issn><issn>1573-6881</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2002</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><recordid>eNpdkEtLw0AUhQdRbK2u3Ulw4S46j2Qe7krxBQVFFNyFyeQOnZJk4kwC1l9vbOvG1YFzv3u45yJ0TvA1wZTdzG9HoTlWCos8ZwdoSnLBUi4lOURTTLI8zYT6mKCTGNcYY4lzfIwmhHIhM8amCF6G4Kwzune-TXRbJWalgzY9BPe9M71N-hUkxjddDV_JU2KDb7ZWgNi5oHsfNr9rbsu-rnzlx2lohpg0Orh2iKfoyOo6wtleZ-j9_u5t8Zgunx-eFvNlaqjEfcq5KkEbpqmmwIwtCSGWgNBcaJIzyQGsVkYCcGZUVjEhdSWAUCCCKFuyGbra5XbBfw4Q-6Jx0UBd6xb8EAtBJWeZ4iN4-Q9c-yG0422FyAUhGGdqhC720FA2UBVdcGOfTfH3PfYDSYJzFQ</recordid><startdate>200212</startdate><enddate>200212</enddate><creator>Fernandes, Andreia S</creator><creator>Pereira, Manuela M</creator><creator>Teixeira, Miguel</creator><general>Springer Nature B.V</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>3V.</scope><scope>7QO</scope><scope>7QP</scope><scope>7TK</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M2P</scope><scope>M7P</scope><scope>P64</scope><scope>PDBOC</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>7X8</scope></search><sort><creationdate>200212</creationdate><title>Purification and characterization of the complex I from the respiratory chain of Rhodothermus marinus</title><author>Fernandes, Andreia S ; Pereira, Manuela M ; Teixeira, Miguel</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c280t-669beac3a2a2e3cfb111f1e7a67a15386eefa9c8ee63c94d378ad7e12e1719fb3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2002</creationdate><topic>Bacteria</topic><topic>Electron Spin Resonance Spectroscopy</topic><topic>Electron Transport Complex I</topic><topic>Enzymes</topic><topic>Gram-Negative Aerobic Bacteria - enzymology</topic><topic>Kinetics</topic><topic>Liposomes</topic><topic>Molecular Weight</topic><topic>NAD - metabolism</topic><topic>NADH, NADPH Oxidoreductases - chemistry</topic><topic>NADH, NADPH Oxidoreductases - isolation & purification</topic><topic>NADH, NADPH Oxidoreductases - metabolism</topic><topic>Protein Subunits</topic><topic>Rotenone</topic><topic>Spectrometry, Fluorescence</topic><topic>Spectrophotometry</topic><topic>Spectroscopy</topic><topic>Sulfur</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Fernandes, Andreia S</creatorcontrib><creatorcontrib>Pereira, Manuela M</creatorcontrib><creatorcontrib>Teixeira, Miguel</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>ProQuest Central (Corporate)</collection><collection>Biotechnology Research Abstracts</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Materials Science & Engineering Collection</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Technology Collection</collection><collection>Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Materials Science Collection</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Materials Science Database</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Science Database</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Materials Science Collection</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central Basic</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of bioenergetics and biomembranes</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Fernandes, Andreia S</au><au>Pereira, Manuela M</au><au>Teixeira, Miguel</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Purification and characterization of the complex I from the respiratory chain of Rhodothermus marinus</atitle><jtitle>Journal of bioenergetics and biomembranes</jtitle><addtitle>J Bioenerg Biomembr</addtitle><date>2002-12</date><risdate>2002</risdate><volume>34</volume><issue>6</issue><spage>413</spage><epage>421</epage><pages>413-421</pages><issn>0145-479X</issn><eissn>1573-6881</eissn><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.</abstract><cop>United States</cop><pub>Springer Nature B.V</pub><pmid>12678433</pmid><doi>10.1023/A:1022509907553</doi><tpages>9</tpages></addata></record> |
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| subjects | Bacteria Electron Spin Resonance Spectroscopy Electron Transport Complex I Enzymes Gram-Negative Aerobic Bacteria - enzymology Kinetics Liposomes Molecular Weight NAD - metabolism NADH, NADPH Oxidoreductases - chemistry NADH, NADPH Oxidoreductases - isolation & purification NADH, NADPH Oxidoreductases - metabolism Protein Subunits Rotenone Spectrometry, Fluorescence Spectrophotometry Spectroscopy Sulfur |
| title | Purification and characterization of the complex I from the respiratory chain of Rhodothermus marinus |
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