Fluorescent
Probes for Tracking the Transfer of Iron–Sulfur
Cluster and Other Metal Cofactors in Biosynthetic Reaction Pathways
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Abstract
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. We anticipate
that this cluster detection methodology will transform the study of
Fe–S cluster pathways and potentially other metal cofactor
biosynthetic pathways