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

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.

Schizosaccharomyces pombe Grx4 regulates the transcriptional repressor Php4 via [2Fe–2S] cluster binding

“…4A), suggesting that Php4 does not bind the cluster or alter the Fe-S cluster coordination environment under these conditions. The addition of 5 mM DTT to this mixture, which has been shown to accelerate Fe-S cluster transfer reactions, 31 also did not facilitate formation of the [2Fe-2S] Php4-Grx4 heterocomplex (data not shown). These results demonstrate that [2Fe-2S] Grx4 cannot be converted to the [2Fe-2S] Php4-Grx4 complex by simple titration with Php4.…”

Iron–sulfur cluster exchange reactions mediated by the human Nfu protein

“…Recent studies have suggested formation of Fe–S cluster-associated high molecular weight species (HMWS) when excess DTT is added to the reaction mixtures after the formation of [2Fe–2S] clusters, which can then either facilitate cluster transfer between putative protein partners or formation of [4Fe–4S] cluster on scaffold proteins [49, 50]. To investigate the role of DTT in our studies, several control reactions were performed by incubating [2Fe–2S](GS) 4 complex, holo Nfu and holo ferredoxins with excess DTT.…”

Mapping cellular Fe–S cluster uptake and exchange reactions – divergent pathways for iron–sulfur cluster delivery to human ferredoxins

“…These can then facilitate cluster transfer between putative protein partners or formation of [4Fe-4S] cluster on scaffold proteins if excess DTT is added after the formation of [2Fe-2S] clusters. 75, 76 In our studies, we incubated holo proteins (holo IscU, holo Isa1, holo Grx2, holo Grx3, holo ferredoxins, and [2Fe-2S](GS 4 ) complex) with excess DTT. No significant change in the CD signal of the holo proteins was observed, clearly indicating that under our conditions, DTT is not forming any intermediate Fe-S species.…”