Cargando…

Cluster-selective (57)Fe labeling of a Twitch-domain-containing radical SAM enzyme

(57)Fe-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 (57)Fe. As a result, it can be unclear...

Descripción completa

Detalles Bibliográficos
Autores principales: Namkoong, Gil, Suess, Daniel L. M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Royal Society of Chemistry 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10337720/
https://www.ncbi.nlm.nih.gov/pubmed/37449070
http://dx.doi.org/10.1039/d3sc02016a
_version_ 1785071475784417280
author Namkoong, Gil
Suess, Daniel L. M.
author_facet Namkoong, Gil
Suess, Daniel L. M.
author_sort Namkoong, Gil
collection PubMed
description (57)Fe-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 (57)Fe. 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 (57)Fe 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 (57)Fe Mössbauer spectroscopic analysis, we show that, of the two [Fe(4)S(4)] 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 Fe(2+); the auxiliary cluster is essentially inert under the reaction conditions. Exploiting this rate difference allows for either of the two [Fe(4)S(4)] clusters to be selectively labeled: the SAM-binding cluster can be labeled by exchanging unlabeled BtrN with (57)Fe(2+), or the auxiliary cluster can be labeled by exchanging fully labeled BtrN with natural abundance Fe(2+). 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.
format Online
Article
Text
id pubmed-10337720
institution National Center for Biotechnology Information
language English
publishDate 2023
publisher The Royal Society of Chemistry
record_format MEDLINE/PubMed
spelling pubmed-103377202023-07-13 Cluster-selective (57)Fe labeling of a Twitch-domain-containing radical SAM enzyme Namkoong, Gil Suess, Daniel L. M. Chem Sci Chemistry (57)Fe-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 (57)Fe. 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 (57)Fe 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 (57)Fe Mössbauer spectroscopic analysis, we show that, of the two [Fe(4)S(4)] 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 Fe(2+); the auxiliary cluster is essentially inert under the reaction conditions. Exploiting this rate difference allows for either of the two [Fe(4)S(4)] clusters to be selectively labeled: the SAM-binding cluster can be labeled by exchanging unlabeled BtrN with (57)Fe(2+), or the auxiliary cluster can be labeled by exchanging fully labeled BtrN with natural abundance Fe(2+). 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. The Royal Society of Chemistry 2023-06-02 /pmc/articles/PMC10337720/ /pubmed/37449070 http://dx.doi.org/10.1039/d3sc02016a Text en This journal is © The Royal Society of Chemistry https://creativecommons.org/licenses/by-nc/3.0/
spellingShingle Chemistry
Namkoong, Gil
Suess, Daniel L. M.
Cluster-selective (57)Fe labeling of a Twitch-domain-containing radical SAM enzyme
title Cluster-selective (57)Fe labeling of a Twitch-domain-containing radical SAM enzyme
title_full Cluster-selective (57)Fe labeling of a Twitch-domain-containing radical SAM enzyme
title_fullStr Cluster-selective (57)Fe labeling of a Twitch-domain-containing radical SAM enzyme
title_full_unstemmed Cluster-selective (57)Fe labeling of a Twitch-domain-containing radical SAM enzyme
title_short Cluster-selective (57)Fe labeling of a Twitch-domain-containing radical SAM enzyme
title_sort cluster-selective (57)fe labeling of a twitch-domain-containing radical sam enzyme
topic Chemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10337720/
https://www.ncbi.nlm.nih.gov/pubmed/37449070
http://dx.doi.org/10.1039/d3sc02016a
work_keys_str_mv AT namkoonggil clusterselective57felabelingofatwitchdomaincontainingradicalsamenzyme
AT suessdaniellm clusterselective57felabelingofatwitchdomaincontainingradicalsamenzyme