Functional Assembly of <i>Caenorhabditis elegans</i> Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase Activity
Among seven homologs of cytochrome <i>b</i><sub>561</sub> in a model organism <i>C. elegans</i>, Cecytb-2 was confirmed to be expressed in digestive organs and was considered as a homolog of human Dcytb functioning as a ferric reductase. Cecytb-2 protein was expre...
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2021-01-01
|
| Series: | Biomolecules |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2218-273X/11/1/96 |
| _version_ | 1797412054266544128 |
|---|---|
| author | Hamed A. Abosharaf Yuki Sakamoto Aliaa M. Radwan Keisuke Yuzu Mika Fujimura Thoria Diab Tarek M. Mohamed Eri Chatani Tetsunari Kimura Motonari Tsubaki |
| author_facet | Hamed A. Abosharaf Yuki Sakamoto Aliaa M. Radwan Keisuke Yuzu Mika Fujimura Thoria Diab Tarek M. Mohamed Eri Chatani Tetsunari Kimura Motonari Tsubaki |
| author_sort | Hamed A. Abosharaf |
| collection | DOAJ |
| description | Among seven homologs of cytochrome <i>b</i><sub>561</sub> in a model organism <i>C. elegans</i>, Cecytb-2 was confirmed to be expressed in digestive organs and was considered as a homolog of human Dcytb functioning as a ferric reductase. Cecytb-2 protein was expressed in <i>Pichia pastoris</i> cells, purified, and reconstituted into a phospholipid bilayer nanodisc. The reconstituted Cecytb-2 in nanodisc environments was extremely stable and more reducible with ascorbate than in a detergent-micelle state. We confirmed the ferric reductase activity of Cecytb-2 by analyzing the oxidation of ferrous heme upon addition of ferric substrate under anaerobic conditions, where clear and saturable dependencies on the substrate concentrations following the Michaelis–Menten equation were observed. Further, we confirmed that the ferric substrate was converted to a ferrous state by using a nitroso-PSAP assay. Importantly, we observed that the ferric reductase activity of Cecytb-2 became enhanced in the phospholipid bilayer nanodisc. |
| first_indexed | 2024-03-09T04:56:32Z |
| format | Article |
| id | doaj.art-c5e71f0d8e1b4611885d821be88adf5a |
| institution | Directory Open Access Journal |
| issn | 2218-273X |
| language | English |
| last_indexed | 2024-03-09T04:56:32Z |
| publishDate | 2021-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomolecules |
| spelling | doaj.art-c5e71f0d8e1b4611885d821be88adf5a2023-12-03T13:05:51ZengMDPI AGBiomolecules2218-273X2021-01-011119610.3390/biom11010096Functional Assembly of <i>Caenorhabditis elegans</i> Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase ActivityHamed A. Abosharaf0Yuki Sakamoto1Aliaa M. Radwan2Keisuke Yuzu3Mika Fujimura4Thoria Diab5Tarek M. Mohamed6Eri Chatani7Tetsunari Kimura8Motonari Tsubaki9Department of Chemistry, Graduate School of Science, Kobe University, Nada-ku, Kobe, Hyogo 657-8501, JapanDepartment of Chemistry, Graduate School of Science, Kobe University, Nada-ku, Kobe, Hyogo 657-8501, JapanDepartment of Chemistry, Graduate School of Science, Kobe University, Nada-ku, Kobe, Hyogo 657-8501, JapanDepartment of Chemistry, Graduate School of Science, Kobe University, Nada-ku, Kobe, Hyogo 657-8501, JapanDepartment of Chemistry, Graduate School of Science, Kobe University, Nada-ku, Kobe, Hyogo 657-8501, JapanBiochemistry Division, Chemistry Department, Faculty of Science, Tanta University, Tanta 31527, EgyptBiochemistry Division, Chemistry Department, Faculty of Science, Tanta University, Tanta 31527, EgyptDepartment of Chemistry, Graduate School of Science, Kobe University, Nada-ku, Kobe, Hyogo 657-8501, JapanDepartment of Chemistry, Graduate School of Science, Kobe University, Nada-ku, Kobe, Hyogo 657-8501, JapanDepartment of Chemistry, Graduate School of Science, Kobe University, Nada-ku, Kobe, Hyogo 657-8501, JapanAmong seven homologs of cytochrome <i>b</i><sub>561</sub> in a model organism <i>C. elegans</i>, Cecytb-2 was confirmed to be expressed in digestive organs and was considered as a homolog of human Dcytb functioning as a ferric reductase. Cecytb-2 protein was expressed in <i>Pichia pastoris</i> cells, purified, and reconstituted into a phospholipid bilayer nanodisc. The reconstituted Cecytb-2 in nanodisc environments was extremely stable and more reducible with ascorbate than in a detergent-micelle state. We confirmed the ferric reductase activity of Cecytb-2 by analyzing the oxidation of ferrous heme upon addition of ferric substrate under anaerobic conditions, where clear and saturable dependencies on the substrate concentrations following the Michaelis–Menten equation were observed. Further, we confirmed that the ferric substrate was converted to a ferrous state by using a nitroso-PSAP assay. Importantly, we observed that the ferric reductase activity of Cecytb-2 became enhanced in the phospholipid bilayer nanodisc.https://www.mdpi.com/2218-273X/11/1/96<i>C. elegans</i>Cecytb-2ferric reductasenanodiscnitroso-PSAP |
| spellingShingle | Hamed A. Abosharaf Yuki Sakamoto Aliaa M. Radwan Keisuke Yuzu Mika Fujimura Thoria Diab Tarek M. Mohamed Eri Chatani Tetsunari Kimura Motonari Tsubaki Functional Assembly of <i>Caenorhabditis elegans</i> Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase Activity Biomolecules <i>C. elegans</i> Cecytb-2 ferric reductase nanodisc nitroso-PSAP |
| title | Functional Assembly of <i>Caenorhabditis elegans</i> Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase Activity |
| title_full | Functional Assembly of <i>Caenorhabditis elegans</i> Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase Activity |
| title_fullStr | Functional Assembly of <i>Caenorhabditis elegans</i> Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase Activity |
| title_full_unstemmed | Functional Assembly of <i>Caenorhabditis elegans</i> Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase Activity |
| title_short | Functional Assembly of <i>Caenorhabditis elegans</i> Cytochrome b-2 (Cecytb-2) into Phospholipid Bilayer Nanodisc with Enhanced Iron Reductase Activity |
| title_sort | functional assembly of i caenorhabditis elegans i cytochrome b 2 cecytb 2 into phospholipid bilayer nanodisc with enhanced iron reductase activity |
| topic | <i>C. elegans</i> Cecytb-2 ferric reductase nanodisc nitroso-PSAP |
| url | https://www.mdpi.com/2218-273X/11/1/96 |
| work_keys_str_mv | AT hamedaabosharaf functionalassemblyoficaenorhabditiselegansicytochromeb2cecytb2intophospholipidbilayernanodiscwithenhancedironreductaseactivity AT yukisakamoto functionalassemblyoficaenorhabditiselegansicytochromeb2cecytb2intophospholipidbilayernanodiscwithenhancedironreductaseactivity AT aliaamradwan functionalassemblyoficaenorhabditiselegansicytochromeb2cecytb2intophospholipidbilayernanodiscwithenhancedironreductaseactivity AT keisukeyuzu functionalassemblyoficaenorhabditiselegansicytochromeb2cecytb2intophospholipidbilayernanodiscwithenhancedironreductaseactivity AT mikafujimura functionalassemblyoficaenorhabditiselegansicytochromeb2cecytb2intophospholipidbilayernanodiscwithenhancedironreductaseactivity AT thoriadiab functionalassemblyoficaenorhabditiselegansicytochromeb2cecytb2intophospholipidbilayernanodiscwithenhancedironreductaseactivity AT tarekmmohamed functionalassemblyoficaenorhabditiselegansicytochromeb2cecytb2intophospholipidbilayernanodiscwithenhancedironreductaseactivity AT erichatani functionalassemblyoficaenorhabditiselegansicytochromeb2cecytb2intophospholipidbilayernanodiscwithenhancedironreductaseactivity AT tetsunarikimura functionalassemblyoficaenorhabditiselegansicytochromeb2cecytb2intophospholipidbilayernanodiscwithenhancedironreductaseactivity AT motonaritsubaki functionalassemblyoficaenorhabditiselegansicytochromeb2cecytb2intophospholipidbilayernanodiscwithenhancedironreductaseactivity |