Cluster-selective 57Fe labeling of a Twitch-domaincontaining radical SAM enzyme
57Fe-specific techniques such as Mössbauer spectroscopy are invaluable tools in mechanistic studies of Fe–S proteins. However, they remain underutilized for proteins that bind multiple Fe–S clusters because such proteins are typically uniformly enriched with 57Fe. As a result, it can be unclear whic...
| Main Authors: | , |
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| Other Authors: | |
| Format: | Article |
| Language: | English |
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Royal Society of Chemistry
2024
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| Online Access: | https://hdl.handle.net/1721.1/156920 |
| _version_ | 1811084266064314368 |
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| author | Namkoong, Gil Suess, Daniel LM |
| author2 | Massachusetts Institute of Technology. Department of Chemistry |
| author_facet | Massachusetts Institute of Technology. Department of Chemistry Namkoong, Gil Suess, Daniel LM |
| author_sort | Namkoong, Gil |
| collection | MIT |
| description | 57Fe-specific techniques such as Mössbauer spectroscopy are invaluable tools in mechanistic studies of Fe–S proteins. However, they remain underutilized for proteins that bind multiple Fe–S clusters because such proteins are typically uniformly enriched with 57Fe. As a result, it can be unclear which spectroscopic responses derive from which cluster, and this in turn obscures the chemistry that takes place at each cluster. Herein, we report a facile method for cluster-selective 57Fe enrichment based on exchange between the protein's Fe–S clusters and exogenous Fe ions. Through a combination of inductively coupled plasma mass spectrometric and 57Fe Mössbauer spectroscopic analysis, we show that, of the two [Fe4S4] clusters in BtrN (a Twitch-domain-containing radical S-adenosyl-L-methionine (SAM) enzyme), the Fe ions in the SAM-binding cluster undergo faster exchange with exogenous Fe2+; the auxiliary cluster is essentially inert under the reaction conditions. Exploiting this rate difference allows for either of the two [Fe4S4] clusters to be selectively labeled: the SAM-binding cluster can be labeled by exchanging unlabeled BtrN with 57Fe2+, or the auxiliary cluster can be labeled by exchanging fully labeled BtrN with natural abundance Fe2+. The labeling selectivity likely originates primarily from differences in the clusters' accessibility to small molecules, with secondary contributions from the different redox properties of the clusters. This method for cluster-selective isotopic labeling could in principle be applied to any protein that binds multiple Fe–S clusters so long as the clusters undergo exchange with exogenous Fe ions at sufficiently different rates. |
| first_indexed | 2024-09-23T12:47:59Z |
| format | Article |
| id | mit-1721.1/156920 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T12:47:59Z |
| publishDate | 2024 |
| publisher | Royal Society of Chemistry |
| record_format | dspace |
| spelling | mit-1721.1/1569202024-09-21T03:31:24Z Cluster-selective 57Fe labeling of a Twitch-domaincontaining radical SAM enzyme Namkoong, Gil Suess, Daniel LM Massachusetts Institute of Technology. Department of Chemistry 57Fe-specific techniques such as Mössbauer spectroscopy are invaluable tools in mechanistic studies of Fe–S proteins. However, they remain underutilized for proteins that bind multiple Fe–S clusters because such proteins are typically uniformly enriched with 57Fe. As a result, it can be unclear which spectroscopic responses derive from which cluster, and this in turn obscures the chemistry that takes place at each cluster. Herein, we report a facile method for cluster-selective 57Fe enrichment based on exchange between the protein's Fe–S clusters and exogenous Fe ions. Through a combination of inductively coupled plasma mass spectrometric and 57Fe Mössbauer spectroscopic analysis, we show that, of the two [Fe4S4] clusters in BtrN (a Twitch-domain-containing radical S-adenosyl-L-methionine (SAM) enzyme), the Fe ions in the SAM-binding cluster undergo faster exchange with exogenous Fe2+; the auxiliary cluster is essentially inert under the reaction conditions. Exploiting this rate difference allows for either of the two [Fe4S4] clusters to be selectively labeled: the SAM-binding cluster can be labeled by exchanging unlabeled BtrN with 57Fe2+, or the auxiliary cluster can be labeled by exchanging fully labeled BtrN with natural abundance Fe2+. The labeling selectivity likely originates primarily from differences in the clusters' accessibility to small molecules, with secondary contributions from the different redox properties of the clusters. This method for cluster-selective isotopic labeling could in principle be applied to any protein that binds multiple Fe–S clusters so long as the clusters undergo exchange with exogenous Fe ions at sufficiently different rates. 2024-09-20T17:57:42Z 2024-09-20T17:57:42Z 2023 2024-09-20T17:06:15Z Article http://purl.org/eprint/type/JournalArticle https://hdl.handle.net/1721.1/156920 Chem. Sci., 2023,14, 7492-7499 en 10.1039/d3sc02016a Chemical Science Creative Commons Attribution-Noncommercial http://creativecommons.org/licenses/by-nc/3.0/ application/pdf Royal Society of Chemistry Royal Society of Chemistry |
| spellingShingle | Namkoong, Gil Suess, Daniel LM Cluster-selective 57Fe labeling of a Twitch-domaincontaining radical SAM enzyme |
| title | Cluster-selective 57Fe labeling of a Twitch-domaincontaining radical SAM enzyme |
| title_full | Cluster-selective 57Fe labeling of a Twitch-domaincontaining radical SAM enzyme |
| title_fullStr | Cluster-selective 57Fe labeling of a Twitch-domaincontaining radical SAM enzyme |
| title_full_unstemmed | Cluster-selective 57Fe labeling of a Twitch-domaincontaining radical SAM enzyme |
| title_short | Cluster-selective 57Fe labeling of a Twitch-domaincontaining radical SAM enzyme |
| title_sort | cluster selective 57fe labeling of a twitch domaincontaining radical sam enzyme |
| url | https://hdl.handle.net/1721.1/156920 |
| work_keys_str_mv | AT namkoonggil clusterselective57felabelingofatwitchdomaincontainingradicalsamenzyme AT suessdaniellm clusterselective57felabelingofatwitchdomaincontainingradicalsamenzyme |