Fluorescent Probes for Tracking the Transfer of Iron–Sulfur Cluster and Other Metal Cofactors in Biosynthetic Reaction Pathways

Vranish, James N.; Russell, William K.; Yu, Lusa E.; Cox, Rachael M.; Russell, David H.; Barondeau, David P.

doi:10.1021/ja510998s

Title: Fluorescent Probes for Tracking the Transfer of Iron–Sulfur Cluster and Other Metal Cofactors in Biosynthetic Reaction Pathways

Journal Article · Wed Dec 24 00:00:00 EST 2014 · Journal of the American Chemical Society

DOI:https://doi.org/10.1021/ja510998s· OSTI ID:1167284

Vranish, James N. ^[1]; Russell, William K. ^[1]; Yu, Lusa E. ^[1]; Cox, Rachael M. ^[1]; Russell, David H. ^[1]; Barondeau, David P. ^[1]

Department of Biochemistry and Biophysics and ‡Department of Chemistry, Texas A&M University, College Station, Texas 77842-3012, United States

Iron–sulfur (Fe–S) clusters are protein cofactors that are constructed and delivered to target proteins by elaborate biosynthetic machinery. Mechanistic insights into these processes have been limited by the lack of sensitive probes for tracking Fe–S cluster synthesis and transfer reactions. Here we present fusion protein- and intein-based fluorescent labeling strategies that can probe Fe–S cluster binding. The fluorescence is sensitive to different cluster types ([2Fe–2S] and [4Fe–4S] clusters), ligand environments ([2Fe–2S] clusters on Rieske, ferredoxin (Fdx), and glutaredoxin), and cluster oxidation states. The power of this approach is highlighted with an extreme example in which the kinetics of Fe–S cluster transfer reactions are monitored between two Fdx molecules that have identical Fe–S spectroscopic properties. This exchange reaction between labeled and unlabeled Fdx is catalyzed by dithiothreitol (DTT), a result that was confirmed by mass spectrometry. DTT likely functions in a ligand substitution reaction that generates a [2Fe–2S]–DTT species, which can transfer the cluster to either labeled or unlabeled Fdx. The ability to monitor this challenging cluster exchange reaction indicates that real-time Fe–S cluster incorporation can be tracked for a specific labeled protein in multicomponent assays that include several unlabeled Fe–S binding proteins or other chromophores. Such advanced kinetic experiments are required to untangle the intricate networks of transfer pathways and the factors affecting flux through branch points. High sensitivity and suitability with high-throughput methodology are additional benefits of this approach. Lastly, we anticipate that this cluster detection methodology will transform the study of Fe–S cluster pathways and potentially other metal cofactor biosynthetic pathways.

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Research Organization:: Texas A&M Univ., College Station, TX (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES)

Grant/Contract Number:: FG02-04ER15520

OSTI ID:: 1167284

Alternate ID(s):: OSTI ID: 1346169

Journal Information:: Journal of the American Chemical Society, Journal Name: Journal of the American Chemical Society Vol. 137 Journal Issue: 1; ISSN 0002-7863

Publisher:: American Chemical SocietyCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 19 works

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