Measuring the Cytochrome c Nitrite Reductase Activity—Practical Considerations on the Enzyme Assays
The cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desulfuricans ATCC 27774 is able to reduce nitrite to ammonia in a six-electron transfer reaction. Although extensively characterized from the spectroscopic and structural points-of-view, some of its kinetic aspects are still under explor...
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| Format: | Article |
| Language: | English |
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Wiley
2010-01-01
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| Series: | Bioinorganic Chemistry and Applications |
| Online Access: | http://dx.doi.org/10.1155/2010/634597 |
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| author | Célia M. Silveira Stéphane Besson Isabel Moura José J. G. Moura M. Gabriela Almeida |
| author_facet | Célia M. Silveira Stéphane Besson Isabel Moura José J. G. Moura M. Gabriela Almeida |
| author_sort | Célia M. Silveira |
| collection | DOAJ |
| description | The cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desulfuricans ATCC 27774 is
able to reduce nitrite to ammonia in a six-electron transfer reaction. Although extensively
characterized from the spectroscopic and structural points-of-view, some of its kinetic aspects
are still under explored. In this work the kinetic behaviour of ccNiR has been evaluated in a
systematic manner using two different spectrophotometric assays carried out in the presence of
different redox mediators and a direct electrochemical approach. Solution assays have proved
that the specific activity of ccNiR decreases with the reduction potential of the electronic carriers
and ammonium is always the main product of nitrite reduction. The catalytic parameters were
discussed on the basis of the mediator reducing power and also taking into account the location
of their putative docking sites with ccNiR. Due to the fast kinetics of ccNiR, electron delivering
from reduced electron donors is rate-limiting in all spectrophotometric assays, so the estimated
kinetic constants are apparent only. Nevertheless, this limitation could be overcome by using a
direct electrochemical approach which shows that the binding affinity for nitrite decreases whilst
turnover increases with the reductive driving force. |
| format | Article |
| id | doaj-art-ad9cbe474ca24d4faeecf24803a09eed |
| institution | Kabale University |
| issn | 1565-3633 1687-479X |
| language | English |
| publishDate | 2010-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | Bioinorganic Chemistry and Applications |
| spelling | doaj-art-ad9cbe474ca24d4faeecf24803a09eed2025-02-03T01:12:01ZengWileyBioinorganic Chemistry and Applications1565-36331687-479X2010-01-01201010.1155/2010/634597634597Measuring the Cytochrome c Nitrite Reductase Activity—Practical Considerations on the Enzyme AssaysCélia M. Silveira0Stéphane Besson1Isabel Moura2José J. G. Moura3M. Gabriela Almeida4REQUIMTE, Departamento de Química, CQFB, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, PortugalREQUIMTE, Departamento de Química, CQFB, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, PortugalREQUIMTE, Departamento de Química, CQFB, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, PortugalREQUIMTE, Departamento de Química, CQFB, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, PortugalREQUIMTE, Departamento de Química, CQFB, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, PortugalThe cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desulfuricans ATCC 27774 is able to reduce nitrite to ammonia in a six-electron transfer reaction. Although extensively characterized from the spectroscopic and structural points-of-view, some of its kinetic aspects are still under explored. In this work the kinetic behaviour of ccNiR has been evaluated in a systematic manner using two different spectrophotometric assays carried out in the presence of different redox mediators and a direct electrochemical approach. Solution assays have proved that the specific activity of ccNiR decreases with the reduction potential of the electronic carriers and ammonium is always the main product of nitrite reduction. The catalytic parameters were discussed on the basis of the mediator reducing power and also taking into account the location of their putative docking sites with ccNiR. Due to the fast kinetics of ccNiR, electron delivering from reduced electron donors is rate-limiting in all spectrophotometric assays, so the estimated kinetic constants are apparent only. Nevertheless, this limitation could be overcome by using a direct electrochemical approach which shows that the binding affinity for nitrite decreases whilst turnover increases with the reductive driving force.http://dx.doi.org/10.1155/2010/634597 |
| spellingShingle | Célia M. Silveira Stéphane Besson Isabel Moura José J. G. Moura M. Gabriela Almeida Measuring the Cytochrome c Nitrite Reductase Activity—Practical Considerations on the Enzyme Assays Bioinorganic Chemistry and Applications |
| title | Measuring the Cytochrome c Nitrite Reductase Activity—Practical Considerations on the Enzyme Assays |
| title_full | Measuring the Cytochrome c Nitrite Reductase Activity—Practical Considerations on the Enzyme Assays |
| title_fullStr | Measuring the Cytochrome c Nitrite Reductase Activity—Practical Considerations on the Enzyme Assays |
| title_full_unstemmed | Measuring the Cytochrome c Nitrite Reductase Activity—Practical Considerations on the Enzyme Assays |
| title_short | Measuring the Cytochrome c Nitrite Reductase Activity—Practical Considerations on the Enzyme Assays |
| title_sort | measuring the cytochrome c nitrite reductase activity practical considerations on the enzyme assays |
| url | http://dx.doi.org/10.1155/2010/634597 |
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