Voltammetric studies of the catalytic mechanism of the respiratory nitrate reductase from Escherichia coli: how nitrate reduction and inhibition depend on the oxidation state of the active site.
The respiratory molybdoenzyme nitrate reductase (NarGHI) from Escherichia coli has been studied by protein film voltammetry, with the enzyme adsorbed on a rotating disk pyrolytic graphite edge (PGE) electrode. Catalytic voltammograms for nitrate reduction show a complex wave consisting of two compon...
| Main Authors: | , , , , , , |
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| Format: | Journal article |
| Language: | English |
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2004
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| _version_ | 1797064969857007616 |
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| author | Elliott, S Hoke, K Heffron, K Palak, M Rothery, R Weiner, J Armstrong, F |
| author_facet | Elliott, S Hoke, K Heffron, K Palak, M Rothery, R Weiner, J Armstrong, F |
| author_sort | Elliott, S |
| collection | OXFORD |
| description | The respiratory molybdoenzyme nitrate reductase (NarGHI) from Escherichia coli has been studied by protein film voltammetry, with the enzyme adsorbed on a rotating disk pyrolytic graphite edge (PGE) electrode. Catalytic voltammograms for nitrate reduction show a complex wave consisting of two components that vary with pH, nitrate concentration, and the presence of inhibitors. At micromolar levels of nitrate, the activity reaches a maximum value at approximately -25 mV and then decreases as the potential becomes more negative. As the nitrate concentration is raised, the activity at more negative potentials increases and eventually becomes the dominant feature at millimolar concentrations. This leads to the hypothesis that nitrate binds more tightly to Mo(V) than Mo(IV), so that low levels of nitrate are more effectively reduced at a higher potential despite the lower driving force. However, an alternative interpretation, that nitrate binding is affected by a change in the redox state of the pterin, cannot be ruled out. This proposal, implicating a specific redox transition at the active site, is supported by experiments carried out using the inhibitors azide and thiocyanate. Azide is the stronger inhibitor of the two, and each inhibitor shows two inhibition constants, one at high potential and one at low potential, both of which are fully competitive with nitrate; closer analysis reveals that the inhibitors act preferentially upon the catalytic activity at high potential. The unusual potential dependence therefore derives from the weaker binding of nitrate or the inhibitors to a more reduced state of the active site. The possible manifestation of these characteristics in vivo has interesting implications for the bioenergetics of E. coli. |
| first_indexed | 2024-03-06T21:21:55Z |
| format | Journal article |
| id | oxford-uuid:41bac485-609f-4675-97c3-4a41754d7727 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T21:21:55Z |
| publishDate | 2004 |
| record_format | dspace |
| spelling | oxford-uuid:41bac485-609f-4675-97c3-4a41754d77272022-03-26T14:45:27ZVoltammetric studies of the catalytic mechanism of the respiratory nitrate reductase from Escherichia coli: how nitrate reduction and inhibition depend on the oxidation state of the active site.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:41bac485-609f-4675-97c3-4a41754d7727EnglishSymplectic Elements at Oxford2004Elliott, SHoke, KHeffron, KPalak, MRothery, RWeiner, JArmstrong, FThe respiratory molybdoenzyme nitrate reductase (NarGHI) from Escherichia coli has been studied by protein film voltammetry, with the enzyme adsorbed on a rotating disk pyrolytic graphite edge (PGE) electrode. Catalytic voltammograms for nitrate reduction show a complex wave consisting of two components that vary with pH, nitrate concentration, and the presence of inhibitors. At micromolar levels of nitrate, the activity reaches a maximum value at approximately -25 mV and then decreases as the potential becomes more negative. As the nitrate concentration is raised, the activity at more negative potentials increases and eventually becomes the dominant feature at millimolar concentrations. This leads to the hypothesis that nitrate binds more tightly to Mo(V) than Mo(IV), so that low levels of nitrate are more effectively reduced at a higher potential despite the lower driving force. However, an alternative interpretation, that nitrate binding is affected by a change in the redox state of the pterin, cannot be ruled out. This proposal, implicating a specific redox transition at the active site, is supported by experiments carried out using the inhibitors azide and thiocyanate. Azide is the stronger inhibitor of the two, and each inhibitor shows two inhibition constants, one at high potential and one at low potential, both of which are fully competitive with nitrate; closer analysis reveals that the inhibitors act preferentially upon the catalytic activity at high potential. The unusual potential dependence therefore derives from the weaker binding of nitrate or the inhibitors to a more reduced state of the active site. The possible manifestation of these characteristics in vivo has interesting implications for the bioenergetics of E. coli. |
| spellingShingle | Elliott, S Hoke, K Heffron, K Palak, M Rothery, R Weiner, J Armstrong, F Voltammetric studies of the catalytic mechanism of the respiratory nitrate reductase from Escherichia coli: how nitrate reduction and inhibition depend on the oxidation state of the active site. |
| title | Voltammetric studies of the catalytic mechanism of the respiratory nitrate reductase from Escherichia coli: how nitrate reduction and inhibition depend on the oxidation state of the active site. |
| title_full | Voltammetric studies of the catalytic mechanism of the respiratory nitrate reductase from Escherichia coli: how nitrate reduction and inhibition depend on the oxidation state of the active site. |
| title_fullStr | Voltammetric studies of the catalytic mechanism of the respiratory nitrate reductase from Escherichia coli: how nitrate reduction and inhibition depend on the oxidation state of the active site. |
| title_full_unstemmed | Voltammetric studies of the catalytic mechanism of the respiratory nitrate reductase from Escherichia coli: how nitrate reduction and inhibition depend on the oxidation state of the active site. |
| title_short | Voltammetric studies of the catalytic mechanism of the respiratory nitrate reductase from Escherichia coli: how nitrate reduction and inhibition depend on the oxidation state of the active site. |
| title_sort | voltammetric studies of the catalytic mechanism of the respiratory nitrate reductase from escherichia coli how nitrate reduction and inhibition depend on the oxidation state of the active site |
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