Probing the proton channels in subunit N of Complex I from Escherichia coli through intra-subunit cross-linking
Copyright © 2016 Elsevier B.V. All rights reserved..
Respiratory Complex I appears to have 4 sites for proton translocation, which are coupled to the oxidation of NADH and reduction of coenzyme Q. The proton pathways are thought to be made of offset half-channels that connect to the membrane surfaces, and are connected by a horizontal path through the center of the membrane. In this study of the enzyme from Escherichia coli, subunit N, containing one of the sites, was targeted. Pairs of cysteine residues were introduced into neighboring α-helices along the proposed proton pathways. In an effort to constrain conformational changes that might occur during proton translocation, we attempted to form disulfide bonds or methanethiosulfonate bridges between two engineered cysteine residues. Cysteine modification was inferred by the inability of PEG-maleimide to shift the electrophoretic mobility of subunit N, which will occur upon reaction with free sulfhydryl groups. After the cross-linking treatment, NADH oxidase and NADH-driven proton translocation were measured. Ten different pairs of cysteine residues showed evidence of cross-linking. The most significant loss of enzyme activity was seen for residues near the essential Lys 395. This residue is positioned between the proposed proton half-channel to the periplasm and the horizontal connection through subunit N, and is also near the essential Glu 144 of subunit M. The results suggest important conformational changes in this region for the delivery of protons to the periplasm, or for coupling the actions of subunit N to subunit M.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2016 |
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| Erschienen: |
2016 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:1857 |
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| Enthalten in: |
Biochimica et biophysica acta - 1857(2016), 12 vom: 25. Dez., Seite 1840-1848 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Tursun, Ablat [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 07.08.2017 Date Revised 10.12.2019 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1016/j.bbabio.2016.09.005 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM264375831 |
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| 245 | 1 | 0 | |a Probing the proton channels in subunit N of Complex I from Escherichia coli through intra-subunit cross-linking |
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| 500 | |a Date Revised 10.12.2019 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a Copyright © 2016 Elsevier B.V. All rights reserved. | ||
| 520 | |a Respiratory Complex I appears to have 4 sites for proton translocation, which are coupled to the oxidation of NADH and reduction of coenzyme Q. The proton pathways are thought to be made of offset half-channels that connect to the membrane surfaces, and are connected by a horizontal path through the center of the membrane. In this study of the enzyme from Escherichia coli, subunit N, containing one of the sites, was targeted. Pairs of cysteine residues were introduced into neighboring α-helices along the proposed proton pathways. In an effort to constrain conformational changes that might occur during proton translocation, we attempted to form disulfide bonds or methanethiosulfonate bridges between two engineered cysteine residues. Cysteine modification was inferred by the inability of PEG-maleimide to shift the electrophoretic mobility of subunit N, which will occur upon reaction with free sulfhydryl groups. After the cross-linking treatment, NADH oxidase and NADH-driven proton translocation were measured. Ten different pairs of cysteine residues showed evidence of cross-linking. The most significant loss of enzyme activity was seen for residues near the essential Lys 395. This residue is positioned between the proposed proton half-channel to the periplasm and the horizontal connection through subunit N, and is also near the essential Glu 144 of subunit M. The results suggest important conformational changes in this region for the delivery of protons to the periplasm, or for coupling the actions of subunit N to subunit M | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, N.I.H., Extramural | |
| 650 | 4 | |a Complex I | |
| 650 | 4 | |a Cross-linking | |
| 650 | 4 | |a Disulfide | |
| 650 | 4 | |a PEG-maleimide | |
| 650 | 4 | |a Proton channels | |
| 650 | 4 | |a Proton translocation | |
| 650 | 7 | |a Cross-Linking Reagents |2 NLM | |
| 650 | 7 | |a Escherichia coli Proteins |2 NLM | |
| 650 | 7 | |a Maleimides |2 NLM | |
| 650 | 7 | |a Protein Subunits |2 NLM | |
| 650 | 7 | |a Protons |2 NLM | |
| 650 | 7 | |a Ubiquitin |2 NLM | |
| 650 | 7 | |a NAD |2 NLM | |
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| 650 | 7 | |a Polyethylene Glycols |2 NLM | |
| 650 | 7 | |a 3WJQ0SDW1A |2 NLM | |
| 650 | 7 | |a NADH Dehydrogenase |2 NLM | |
| 650 | 7 | |a EC 1.6.99.3 |2 NLM | |
| 650 | 7 | |a Electron Transport Complex I |2 NLM | |
| 650 | 7 | |a EC 7.1.1.2 |2 NLM | |
| 650 | 7 | |a Lysine |2 NLM | |
| 650 | 7 | |a K3Z4F929H6 |2 NLM | |
| 650 | 7 | |a Cysteine |2 NLM | |
| 650 | 7 | |a K848JZ4886 |2 NLM | |
| 700 | 1 | |a Zhu, Shaotong |e verfasserin |4 aut | |
| 700 | 1 | |a Vik, Steven B |e verfasserin |4 aut | |
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| 856 | 4 | 0 | |u http://dx.doi.org/10.1016/j.bbabio.2016.09.005 |3 Volltext |
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