Synchrotron-based Infrared Microanalysis of Biological Redox Processes under Electrochemical Control
We describe a method for addressing redox enzymes adsorbed on a carbon electrode using synchrotron infrared microspectroscopy combined with protein film electrochemistry. Redox enzymes have high turnover frequencies, typically 10-1000 s-1, and therefore fast experimental triggers are needed in order...
| Main Authors: | , , , , , , , , , , , , , , |
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| Format: | Journal article |
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ACS
2016
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| _version_ | 1797074767908438016 |
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| author | Ash, P Reeve, H Vincent, K McPherson, I Nayak, S Quinson, J Hidalgo, R Zhu, T Chung, M Healy, A Lonsdale, T Wehbe, K Kelley, C Frogley, M Cinque, G |
| author_facet | Ash, P Reeve, H Vincent, K McPherson, I Nayak, S Quinson, J Hidalgo, R Zhu, T Chung, M Healy, A Lonsdale, T Wehbe, K Kelley, C Frogley, M Cinque, G |
| author_sort | Ash, P |
| collection | OXFORD |
| description | We describe a method for addressing redox enzymes adsorbed on a carbon electrode using synchrotron infrared microspectroscopy combined with protein film electrochemistry. Redox enzymes have high turnover frequencies, typically 10-1000 s-1, and therefore fast experimental triggers are needed in order to study sub-turnover kinetics and identify the involvement of transient species important to their catalytic mechanism. In an electrochemical experiment this equates to the use of microelectrodes to lower the electrochemical cell constant and enable changes in potential to be applied very rapidly. We use a biological cofactor, flavin mononucleotide, to demonstrate the power of synchrotron infrared microspectroscopy relative to conventional infrared methods and show that vibrational spectra with good signal-to-noise ratios can be collected for adsorbed species with low surface coverages on microelectrodes with a geometric area of 25 × 25 µm2. We then demonstrate the applicability of synchrotron infrared microspectroscopy to adsorbed proteins by reporting potential-induced changes in the flavin mononucleotide active site of a flavoenzyme. The method we describe will allow time-resolved spectroscopic studies of chemical and structural changes at redox sites within a variety of proteins under precise electrochemical control. |
| first_indexed | 2024-03-06T23:40:59Z |
| format | Journal article |
| id | oxford-uuid:6f4b0fc2-e00b-45bd-8de1-a4a9baa40c63 |
| institution | University of Oxford |
| last_indexed | 2024-03-06T23:40:59Z |
| publishDate | 2016 |
| publisher | ACS |
| record_format | dspace |
| spelling | oxford-uuid:6f4b0fc2-e00b-45bd-8de1-a4a9baa40c632022-03-26T19:29:55ZSynchrotron-based Infrared Microanalysis of Biological Redox Processes under Electrochemical ControlJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:6f4b0fc2-e00b-45bd-8de1-a4a9baa40c63Symplectic Elements at OxfordACS2016Ash, PReeve, HVincent, KMcPherson, INayak, SQuinson, JHidalgo, RZhu, TChung, MHealy, ALonsdale, TWehbe, KKelley, CFrogley, MCinque, GWe describe a method for addressing redox enzymes adsorbed on a carbon electrode using synchrotron infrared microspectroscopy combined with protein film electrochemistry. Redox enzymes have high turnover frequencies, typically 10-1000 s-1, and therefore fast experimental triggers are needed in order to study sub-turnover kinetics and identify the involvement of transient species important to their catalytic mechanism. In an electrochemical experiment this equates to the use of microelectrodes to lower the electrochemical cell constant and enable changes in potential to be applied very rapidly. We use a biological cofactor, flavin mononucleotide, to demonstrate the power of synchrotron infrared microspectroscopy relative to conventional infrared methods and show that vibrational spectra with good signal-to-noise ratios can be collected for adsorbed species with low surface coverages on microelectrodes with a geometric area of 25 × 25 µm2. We then demonstrate the applicability of synchrotron infrared microspectroscopy to adsorbed proteins by reporting potential-induced changes in the flavin mononucleotide active site of a flavoenzyme. The method we describe will allow time-resolved spectroscopic studies of chemical and structural changes at redox sites within a variety of proteins under precise electrochemical control. |
| spellingShingle | Ash, P Reeve, H Vincent, K McPherson, I Nayak, S Quinson, J Hidalgo, R Zhu, T Chung, M Healy, A Lonsdale, T Wehbe, K Kelley, C Frogley, M Cinque, G Synchrotron-based Infrared Microanalysis of Biological Redox Processes under Electrochemical Control |
| title | Synchrotron-based Infrared Microanalysis of Biological Redox Processes under Electrochemical Control |
| title_full | Synchrotron-based Infrared Microanalysis of Biological Redox Processes under Electrochemical Control |
| title_fullStr | Synchrotron-based Infrared Microanalysis of Biological Redox Processes under Electrochemical Control |
| title_full_unstemmed | Synchrotron-based Infrared Microanalysis of Biological Redox Processes under Electrochemical Control |
| title_short | Synchrotron-based Infrared Microanalysis of Biological Redox Processes under Electrochemical Control |
| title_sort | synchrotron based infrared microanalysis of biological redox processes under electrochemical control |
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