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.

Figure 1

Fluorescence quenching reports Fe–S cluster binding to labeled proteins. The fluorescence intensity was measured for chemically reconstituted proteins and divided by that of the apoprotein. Error bars (SD) are shown for multiple replicates (n = 3). Key: **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 2

Factors affecting Fdx_Rho fluorescence. The fluorescence of apo- or [2Fe–2S]–Fdx_Rho was measured immediately after the addition of various reagents and plotted relative to a control containing apo-Fdx_Rho. [2Fe–2S] clusters were reconstituted and reduced with dithionite (A) or FldR/NADPH (B). Error bars (SD) are shown for multiple replicates (n = 3). Key: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 3

DTT acceleration of cluster transfer from [2Fe–2S]–Fdx to apo-Fdx_Rho. [2Fe–2S]–Fdx (20 µM) was incubated with apo-Fdx_Rho (1 µM) and 0 (black), 8 (red), or 16 mM (blue) DTT. Three repetitions of each DTT concentration are overlaid. Data were fit as pseudo-first-order reactions in KaleidaGraph (not shown) to determine apparent rates of 0.0013(1) µM cluster min⁻¹ (R² = 0.955) and 0.004 95(4) µM cluster min⁻¹ (R² = 0.998) for the 8 and 16 mM DTT reactions, respectively. The minimum fluorescence was assumed to correspond to 1 µM of transferred cluster. The relationship between the apparent rate constants and DTT concentration suggests a second-order reaction with respect to DTT.

Figure 4

Mass spectrometry revealing DTT-dependent cluster exchange between [2Fe–2S]–Fdx and apo-Fdx_Rho. Mass spectra for the +11 charge species of unlabeled Fdx at the conclusion of cluster transfer reactions for samples (A) lacking DTT, (B) lacking apo-Fdx_Rho, or (C) a complete reaction with [2Fe–2S]–Fdx (80 µM), apo-Fdx_Rho (40 µM), and DTT (20 mM). Deconvolution of m/z peaks identified [2Fe–2S]–Fdx ([M + H] = 12 642.0 Da, expected mass 12 643.7 Da) and apo-Fdx ([M + H] = 12 467.1 Da, expected mass 12 467.9 Da) species. An additional peak in sample B is consistent with apo-Fdx plus two sulfur atoms. (D) Peak intensities for [2Fe–2S]–Fdx and apo-Fdx were integrated for all visible charge states and the percentage of apo-Fdx was plotted for the samples from (A), (B), and (C). The 50% apo-Fdx observed in the presence of DTT and apo-Fdx_Rho agrees well with the expected 41% (assuming [2Fe–2S]–Fdx contained 12% apo-Fdx). Error bars represent a standard error of 4%.

Figure 5

Fdx cluster exchange slowed by reduction. Reactions contained apo-Fdx_Rho (0.5 µM) and [2Fe–2S]–Fdx (10 µM) plus (brown) 20 mM DTT, (cyan) 1 mM sodium dithionite, (pink) 50 nM FldR and 100 µM NADPH, (black) 20 mM DTT and 1 mM sodium dithionite, or (dark blue) 20 mM DTT, 50 nM FldR, and 100 µM NADPH. The plotted data is the average of at least three runs for each sample with the maximum error (SD) for any data point being 0.06 (F_reaction/F_ref)′.

Figure 6

Model of DTT-dependent cluster transfer reaction. DTT initiates a ligand substitution reaction through nucleophilic attack of the [2Fe–2S]²⁺ cluster on Fdx. This forms a DTT–[2Fe–2S]²⁺ cluster species that readily transfers the cluster either back to apo- Fdx or forward to apo-Fdx_Rho, which results in fluorescence quenching.