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Isolation, Characterization and Electron Microscopic Single Particle Analysis of the NADH:Ubiquinone Oxidoreductase (Complex I) from the Hyperthermophilic Eubacterium Aquifex aeolicus
The proton-translocating NADH:ubiquinone oxidoreductase (complex I) has been purified from Aquifex aeolicus, a hyperthermophilic eubacterium of known genome sequence. The purified detergent solubilized enzyme is highly active above 50 °C. The specific activity for electron transfer from NADH to decy...
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Published in: | Biochemistry (Easton) 2003-03, Vol.42 (10), p.3032-3039 |
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description | The proton-translocating NADH:ubiquinone oxidoreductase (complex I) has been purified from Aquifex aeolicus, a hyperthermophilic eubacterium of known genome sequence. The purified detergent solubilized enzyme is highly active above 50 °C. The specific activity for electron transfer from NADH to decylubiquinone is 29 U/mg at 80 °C. The A. aeolicus complex I is completely sensitive to rotenone and 2-n-decyl-quinazoline-4-yl-amine. SDS polyacrylamide gel electrophoresis shows that it may contain up to 14 subunits. N-terminal amino acid sequencing of the bands indicates the presence of a stable subcomplex, which is composed of subunits E, F, and G. The isolated complex is highly stable and active in a temperature range from 50 to 90 °C, with a half-life of about 10 h at 80 °C. The activity shows a linear Arrhenius plot at 50−85 °C with an activation energy at 31.92 J/mol K. Single particle electron microscopy shows that the A. aeolicus complex I has the typical L-shape. However, visual inspection of averaged images reveals many more details in the external arm of the complex than has been observed for complex I from other sources. In addition, the angle (90°) between the cytoplasmic peripheral arm and the membrane intrinsic arm of the complex appears to be invariant. |
doi_str_mv | 10.1021/bi026876v |
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The purified detergent solubilized enzyme is highly active above 50 °C. The specific activity for electron transfer from NADH to decylubiquinone is 29 U/mg at 80 °C. The A. aeolicus complex I is completely sensitive to rotenone and 2-n-decyl-quinazoline-4-yl-amine. SDS polyacrylamide gel electrophoresis shows that it may contain up to 14 subunits. N-terminal amino acid sequencing of the bands indicates the presence of a stable subcomplex, which is composed of subunits E, F, and G. The isolated complex is highly stable and active in a temperature range from 50 to 90 °C, with a half-life of about 10 h at 80 °C. The activity shows a linear Arrhenius plot at 50−85 °C with an activation energy at 31.92 J/mol K. Single particle electron microscopy shows that the A. aeolicus complex I has the typical L-shape. However, visual inspection of averaged images reveals many more details in the external arm of the complex than has been observed for complex I from other sources. In addition, the angle (90°) between the cytoplasmic peripheral arm and the membrane intrinsic arm of the complex appears to be invariant.</description><identifier>ISSN: 0006-2960</identifier><identifier>EISSN: 1520-4995</identifier><identifier>DOI: 10.1021/bi026876v</identifier><identifier>PMID: 12627969</identifier><language>eng</language><publisher>United States: American Chemical Society</publisher><subject>Aquifex aeolicus ; Bacteria - enzymology ; Bacteria - ultrastructure ; Bacterial Proteins - chemistry ; Bacterial Proteins - isolation & purification ; Bacterial Proteins - ultrastructure ; Catalysis ; Chromatography, Gel ; Chromatography, Ion Exchange ; Coloring Agents ; decylubiquinone ; Electron Transport Complex I ; Enzyme Stability ; Hot Temperature ; Image Enhancement ; Microscopy, Electron ; Molybdenum ; NADH, NADPH Oxidoreductases - chemistry ; NADH, NADPH Oxidoreductases - isolation & purification ; NADH, NADPH Oxidoreductases - ultrastructure ; Peptide Fragments - chemistry ; Peptide Fragments - isolation & purification ; Protein Subunits - chemistry ; Protein Subunits - isolation & purification ; Proton Pumps - chemistry ; Proton Pumps - isolation & purification ; Proton Pumps - ultrastructure ; Solubility</subject><ispartof>Biochemistry (Easton), 2003-03, Vol.42 (10), p.3032-3039</ispartof><rights>Copyright © 2003 American Chemical Society</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a380t-210ffc89f8a3b06ceb8d4be85cf8e20b505c2cef99c09ebda21f35769d891fe73</citedby><cites>FETCH-LOGICAL-a380t-210ffc89f8a3b06ceb8d4be85cf8e20b505c2cef99c09ebda21f35769d891fe73</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,27923,27924</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/12627969$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Peng, Guohong</creatorcontrib><creatorcontrib>Fritzsch, Günter</creatorcontrib><creatorcontrib>Zickermann, Volker</creatorcontrib><creatorcontrib>Schägger, Hermann</creatorcontrib><creatorcontrib>Mentele, Reinhardt</creatorcontrib><creatorcontrib>Lottspeich, Friedrich</creatorcontrib><creatorcontrib>Bostina, Mihnea</creatorcontrib><creatorcontrib>Radermacher, Michael</creatorcontrib><creatorcontrib>Huber, Robert</creatorcontrib><creatorcontrib>Stetter, Karl Otto</creatorcontrib><creatorcontrib>Michel, Hartmut</creatorcontrib><title>Isolation, Characterization and Electron Microscopic Single Particle Analysis of the NADH:Ubiquinone Oxidoreductase (Complex I) from the Hyperthermophilic Eubacterium Aquifex aeolicus</title><title>Biochemistry (Easton)</title><addtitle>Biochemistry</addtitle><description>The proton-translocating NADH:ubiquinone oxidoreductase (complex I) has been purified from Aquifex aeolicus, a hyperthermophilic eubacterium of known genome sequence. The purified detergent solubilized enzyme is highly active above 50 °C. The specific activity for electron transfer from NADH to decylubiquinone is 29 U/mg at 80 °C. The A. aeolicus complex I is completely sensitive to rotenone and 2-n-decyl-quinazoline-4-yl-amine. SDS polyacrylamide gel electrophoresis shows that it may contain up to 14 subunits. N-terminal amino acid sequencing of the bands indicates the presence of a stable subcomplex, which is composed of subunits E, F, and G. The isolated complex is highly stable and active in a temperature range from 50 to 90 °C, with a half-life of about 10 h at 80 °C. The activity shows a linear Arrhenius plot at 50−85 °C with an activation energy at 31.92 J/mol K. Single particle electron microscopy shows that the A. aeolicus complex I has the typical L-shape. However, visual inspection of averaged images reveals many more details in the external arm of the complex than has been observed for complex I from other sources. In addition, the angle (90°) between the cytoplasmic peripheral arm and the membrane intrinsic arm of the complex appears to be invariant.</description><subject>Aquifex aeolicus</subject><subject>Bacteria - enzymology</subject><subject>Bacteria - ultrastructure</subject><subject>Bacterial Proteins - chemistry</subject><subject>Bacterial Proteins - isolation & purification</subject><subject>Bacterial Proteins - ultrastructure</subject><subject>Catalysis</subject><subject>Chromatography, Gel</subject><subject>Chromatography, Ion Exchange</subject><subject>Coloring Agents</subject><subject>decylubiquinone</subject><subject>Electron Transport Complex I</subject><subject>Enzyme Stability</subject><subject>Hot Temperature</subject><subject>Image Enhancement</subject><subject>Microscopy, Electron</subject><subject>Molybdenum</subject><subject>NADH, NADPH Oxidoreductases - chemistry</subject><subject>NADH, NADPH Oxidoreductases - isolation & purification</subject><subject>NADH, NADPH Oxidoreductases - ultrastructure</subject><subject>Peptide Fragments - chemistry</subject><subject>Peptide Fragments - isolation & purification</subject><subject>Protein Subunits - chemistry</subject><subject>Protein Subunits - isolation & purification</subject><subject>Proton Pumps - chemistry</subject><subject>Proton Pumps - isolation & purification</subject><subject>Proton Pumps - ultrastructure</subject><subject>Solubility</subject><issn>0006-2960</issn><issn>1520-4995</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2003</creationdate><recordtype>article</recordtype><recordid>eNqFkcFu1DAQhi0EokvhwAsgX0BUImA7iRNzW5alu6LQirYcuFiOM2Zdkji1E7TLi_F6uM2qXJA4zYznm3-s-RF6SslrShh9U1nCeFnwn_fQjOaMJJkQ-X00I4TwhAlODtCjEK5imZEie4gOKOOsEFzM0O91cI0arOte4cVGeaUH8PbX7QtWXY2XDejBx-KT1d4F7Xqr8bntvjeAz5QfrI7JvFPNLtiAncHDBvDn-fvV28vKXo-2cx3g062tnYd61IMKgF8uXNs3sMXrI2y8a29nVrsefEx86_qNbeKW5VhN3xlbPI9SJk4ocLE1hsfogVFNgCf7eIguPywvFqvk5PR4vZifJCotyZAwSozRpTClSivCNVRlnVVQ5tqUwEiVk1wzDUYITQRUtWLUpHnBRV0KaqBID9GLSbf37nqEMMjWBg1NozpwY5BFSgqWltl_QSoYzVKaR_BoAm_OGTwY2XvbKr-TlMgbO-WdnZF9thcdqxbqv-TevwgkE2DDANu7vvI_JC_SIpcXZ-fyy_E7nn399lGmkX8-8UoHeeVGH40L_1j8B5h6unk</recordid><startdate>20030318</startdate><enddate>20030318</enddate><creator>Peng, Guohong</creator><creator>Fritzsch, Günter</creator><creator>Zickermann, Volker</creator><creator>Schägger, Hermann</creator><creator>Mentele, Reinhardt</creator><creator>Lottspeich, Friedrich</creator><creator>Bostina, Mihnea</creator><creator>Radermacher, Michael</creator><creator>Huber, Robert</creator><creator>Stetter, Karl Otto</creator><creator>Michel, Hartmut</creator><general>American Chemical Society</general><scope>BSCLL</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>C1K</scope><scope>F1W</scope><scope>H95</scope><scope>L.G</scope><scope>7X8</scope></search><sort><creationdate>20030318</creationdate><title>Isolation, Characterization and Electron Microscopic Single Particle Analysis of the NADH:Ubiquinone Oxidoreductase (Complex I) from the Hyperthermophilic Eubacterium Aquifex aeolicus</title><author>Peng, Guohong ; Fritzsch, Günter ; Zickermann, Volker ; Schägger, Hermann ; Mentele, Reinhardt ; Lottspeich, Friedrich ; Bostina, Mihnea ; Radermacher, Michael ; Huber, Robert ; Stetter, Karl Otto ; Michel, Hartmut</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a380t-210ffc89f8a3b06ceb8d4be85cf8e20b505c2cef99c09ebda21f35769d891fe73</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2003</creationdate><topic>Aquifex aeolicus</topic><topic>Bacteria - enzymology</topic><topic>Bacteria - ultrastructure</topic><topic>Bacterial Proteins - chemistry</topic><topic>Bacterial Proteins - isolation & purification</topic><topic>Bacterial Proteins - ultrastructure</topic><topic>Catalysis</topic><topic>Chromatography, Gel</topic><topic>Chromatography, Ion Exchange</topic><topic>Coloring Agents</topic><topic>decylubiquinone</topic><topic>Electron Transport Complex I</topic><topic>Enzyme Stability</topic><topic>Hot Temperature</topic><topic>Image Enhancement</topic><topic>Microscopy, Electron</topic><topic>Molybdenum</topic><topic>NADH, NADPH Oxidoreductases - chemistry</topic><topic>NADH, NADPH Oxidoreductases - isolation & purification</topic><topic>NADH, NADPH Oxidoreductases - ultrastructure</topic><topic>Peptide Fragments - chemistry</topic><topic>Peptide Fragments - isolation & purification</topic><topic>Protein Subunits - chemistry</topic><topic>Protein Subunits - isolation & purification</topic><topic>Proton Pumps - chemistry</topic><topic>Proton Pumps - isolation & purification</topic><topic>Proton Pumps - ultrastructure</topic><topic>Solubility</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Peng, Guohong</creatorcontrib><creatorcontrib>Fritzsch, Günter</creatorcontrib><creatorcontrib>Zickermann, Volker</creatorcontrib><creatorcontrib>Schägger, Hermann</creatorcontrib><creatorcontrib>Mentele, Reinhardt</creatorcontrib><creatorcontrib>Lottspeich, Friedrich</creatorcontrib><creatorcontrib>Bostina, Mihnea</creatorcontrib><creatorcontrib>Radermacher, Michael</creatorcontrib><creatorcontrib>Huber, Robert</creatorcontrib><creatorcontrib>Stetter, Karl Otto</creatorcontrib><creatorcontrib>Michel, Hartmut</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ASFA: Aquatic Sciences and Fisheries Abstracts</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) 1: Biological Sciences & Living Resources</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) Professional</collection><collection>MEDLINE - Academic</collection><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Peng, Guohong</au><au>Fritzsch, Günter</au><au>Zickermann, Volker</au><au>Schägger, Hermann</au><au>Mentele, Reinhardt</au><au>Lottspeich, Friedrich</au><au>Bostina, Mihnea</au><au>Radermacher, Michael</au><au>Huber, Robert</au><au>Stetter, Karl Otto</au><au>Michel, Hartmut</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Isolation, Characterization and Electron Microscopic Single Particle Analysis of the NADH:Ubiquinone Oxidoreductase (Complex I) from the Hyperthermophilic Eubacterium Aquifex aeolicus</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>2003-03-18</date><risdate>2003</risdate><volume>42</volume><issue>10</issue><spage>3032</spage><epage>3039</epage><pages>3032-3039</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><abstract>The proton-translocating NADH:ubiquinone oxidoreductase (complex I) has been purified from Aquifex aeolicus, a hyperthermophilic eubacterium of known genome sequence. The purified detergent solubilized enzyme is highly active above 50 °C. The specific activity for electron transfer from NADH to decylubiquinone is 29 U/mg at 80 °C. The A. aeolicus complex I is completely sensitive to rotenone and 2-n-decyl-quinazoline-4-yl-amine. SDS polyacrylamide gel electrophoresis shows that it may contain up to 14 subunits. N-terminal amino acid sequencing of the bands indicates the presence of a stable subcomplex, which is composed of subunits E, F, and G. The isolated complex is highly stable and active in a temperature range from 50 to 90 °C, with a half-life of about 10 h at 80 °C. The activity shows a linear Arrhenius plot at 50−85 °C with an activation energy at 31.92 J/mol K. Single particle electron microscopy shows that the A. aeolicus complex I has the typical L-shape. However, visual inspection of averaged images reveals many more details in the external arm of the complex than has been observed for complex I from other sources. In addition, the angle (90°) between the cytoplasmic peripheral arm and the membrane intrinsic arm of the complex appears to be invariant.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>12627969</pmid><doi>10.1021/bi026876v</doi><tpages>8</tpages></addata></record> |
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subjects | Aquifex aeolicus Bacteria - enzymology Bacteria - ultrastructure Bacterial Proteins - chemistry Bacterial Proteins - isolation & purification Bacterial Proteins - ultrastructure Catalysis Chromatography, Gel Chromatography, Ion Exchange Coloring Agents decylubiquinone Electron Transport Complex I Enzyme Stability Hot Temperature Image Enhancement Microscopy, Electron Molybdenum NADH, NADPH Oxidoreductases - chemistry NADH, NADPH Oxidoreductases - isolation & purification NADH, NADPH Oxidoreductases - ultrastructure Peptide Fragments - chemistry Peptide Fragments - isolation & purification Protein Subunits - chemistry Protein Subunits - isolation & purification Proton Pumps - chemistry Proton Pumps - isolation & purification Proton Pumps - ultrastructure Solubility |
title | Isolation, Characterization and Electron Microscopic Single Particle Analysis of the NADH:Ubiquinone Oxidoreductase (Complex I) from the Hyperthermophilic Eubacterium Aquifex aeolicus |
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