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. 2016 Oct;21(7):825-836.
doi: 10.1007/s00775-016-1381-8. Epub 2016 Aug 18.

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

Christine Wachnowsky #  1   2 , Insiya Fidai #  1   3 , J A Cowan  1   2   3
Affiliations

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

Christine Wachnowsky et al. J Biol Inorg Chem. 2016 Oct.

Abstract

Human Nfu is an iron-sulfur cluster protein that has recently been implicated in multiple mitochondrial dysfunctional syndrome (MMDS1). The Nfu family of proteins shares a highly homologous domain that contains a conserved active site consisting of a CXXC motif. There is less functional conservation between bacterial and human Nfu proteins, particularly concerning their Iron-sulfur cluster binding and transfer roles. Herein, we characterize the cluster exchange chemistry of human Nfu and its capacity to bind and transfer a [2Fe-2S] cluster. The mechanism of cluster uptake from a physiologically relevant [2Fe-2S](GS)4 cluster complex, and extraction of the Nfu-bound iron-sulfur cluster by glutathione are described. Human holo Nfu shows a dimer-tetramer equilibrium with a protein to cluster ratio of 2:1, reflecting the Nfu-bridging [2Fe-2S] cluster. This cluster can be transferred to apo human ferredoxins at relatively fast rates, demonstrating a direct role for human Nfu in the process of [2Fe-2S] cluster trafficking and delivery.

Keywords: Cluster exchange; Iron–sulfur cluster; Metalloprotein; Mitochondrial disease; Nfu.

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Figures

Fig. 1
Fig. 1
UV (a) and CD (b) spectra of [2Fe–2S] cluster-bound Nfu following in vitro reconstitution. All ε and Δε values were calculated based on the monomeric protein concentration
Fig. 2
Fig. 2
Analytical ultracentrifugation profiles of 50 μM Nfu. a Apo Nfu was sedimented in the absence (in black) and the presence (red) of 1 mM TCEP and monitored at 280 nm. Sedimentation profiles were fit to the Lamm equation [26, 27] using a continuous distribution model to obtain the molecular weights shown above the peaks. b Holo Nfu was monitored for sedimentation at 420 nm. The sedimentation data were fit in the same way as the apo protein
Fig. 3
Fig. 3
Determination of second-order rate constants for cluster transfer reactions from [2Fe–2S](GS)4 complex to apo Nfu by use of CD spectroscopy. The decrease in Δε at 363 nm was monitored every 10 s to generate the inset (a), which was fit with an exponential to yield kobs. From kobs, the second-order rate constant was determined based on the [2Fe–2S](GS)4 cluster concentration, yielding a rate constant of 1930 ± 212 M−1 min−1 for cluster uptake. b The initial rate of cluster uptake by 50 μM Nfu at different [2Fe–2S](GS)4 complex concentrations was plotted against cluster concentration, which was varied from 200 to 400 μM, and fit linearly to obtain the second order rate constant which was determined to be 4900 ± 800 M−1 min−1
Fig. 4
Fig. 4
Dependence of the rate of cluster uptake by Nfu on protein concentration. The [2Fe–2S](GS)4 cluster was kept in excess and held constant to maintain pseudo-first order conditions. The concentration of Nfu was varied from 25 to 150 μM, while the cluster concentration was kept constant at 300 μM, and the data was fit to Eq. (5), reflecting inhibition by the dimeric form of the protein. The second-order rate constant determined from the protein dependence for cluster uptake was 2500 ± 1200 M−1 min−1
Fig. 5
Fig. 5
Extraction of the [2Fe–2S] cluster from reconstituted holo human Nfu (10 μM in cluster) to form the [2Fe–2S](GS)4 complex monitored by UV–Vis at 420 nm. Stability of the holo protein was examined in the absence of glutathione as a control (black), while the experimental time-dependence for cluster extraction with a 100-fold excess of GSH is shown in red. For the latter fit, the equation includes both exponential and linear terms, where the linear term accommodates general background cluster degradation, resulting in a second-order rate constant of 130 ± 22 M−1 min−1, with no dependence on glutathione concentration
Fig. 6
Fig. 6
Kinetics of cluster transfer from holo Nfu to apo ferredoxins. a Time course of cluster transfer from holo human Nfu to apo human ferredoxin 1 recorded by CD in 50 mM HEPES, 100 mM NaCl, pH 7.5 at a 1:1 concentration ratio. Spectra were recorded every 2 min after the addition of holo Nfu, and converted to percent cluster transfer b to calculate the second order rate constant of 4695 ± 823 M−1 min−1 based on the concentration of the [2Fe–2S] cluster. c Time course of cluster transfer from holo human Nfu to apo human ferredoxin 2 recorded by CD under the same conditions as for ferredoxin 1. d The CD signal was again converted to the percentage of cluster transferred with time to calculate a second order rate constant of 3849 ± 1242 M−1 min−1
Fig. 7
Fig. 7
Model for [2Fe–2S] cluster uptake by monomeric Nfu, represented by the N- and C-terminal domains, from the [2Fe–2S](GS)4 cluster complex to form an intermediate [2Fe–2S] species with two exogenous GSH ligands. A second monomeric Nfu displaces the GSH molecules to form [2Fe–2S] dimeric Nfu
Fig. 8
Fig. 8
Model for direct [2Fe–2S] cluster uptake by pre-formed dimeric Nfu, shown by the N- and C-terminal domains, from the [2Fe–2S](GS)4 cluster complex
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