Figure 1.
(A) Ribbon diagrams of the soluble parts of NAF-1 (amino acids 57–135) derived from published X-ray analyses (1) (PDB code 3FNV). The protein is homodimeric and is comprised of two main domains – the beta cap and the cluster binding domain, where each protomer contains a [2Fe-2S] cluster (sulfur and iron depicted as yellow and orange spheres, respectively). (B) A magnification of one of the NAF-1 cluster binding sites highlighting the single-coordinating His and three coordinating Cys residues.
Figure 2.
Transfer of NAF-1's [2Fe-2S] clusters to the apo-acceptor protein ferredoxin (apo-Fd).
Upper panel. NAF-1 was incubated at 37°C with a DTT reduced apo-Fd for increasing times (0, 3, 7.5, 15, and 30 minutes) and the products were run on a native gel. The red colored bands are indicative of the [2Fe-2S] cluster in the two proteins. The diagrams to the left show the structure of the [2Fe-2S] protein NAF-1 (upper left) and the structure of the [2Fe-2S] protein Ferredoxin (lower right) (indicated as holo-Fd with the [2Fe-2S] cluster bound). Replacement of single-coordinating His114 in NAF-1 with Cys (H114C) shows no transfer to apo-Fd after 60 min (labeled as H114C 60 min). In addition, holo-Fd was run as a reference (lane on the far right). A Coomassie stain of the native gel directly below shows similar protein levels in the period of experimentation. Lower panel. The percent cluster transfer (CT%) from either wild type NAF-1 or H114C mutated NAF-1 to apo-Fd was determined from UV-Vis absorption spectroscopy data obtained by on-line follow up of the transfer reaction at 458 nm (holo NAF-1 absorbance peak) and at 423 nm (holo-Fd absorbance peak) and calculations as described in experimental procedures. The WT data was fit to an exponential rise as the kinetics display catalytic behavior. The H114C data was fit to a line as the reaction was very slow compared to the WT.
Figure 3.
Transfer from NAF-1 only occurs for the oxidized state of the cluster to the reduced state of apo-Fd.
Upper panel. NAF-1 pre-reduced with dithionite is incapable of transferring its cluster to apo-Fd as analyzed by UV-Vis absorption spectroscopy. Oxidation of the cluster upon addition of oxygen promoted cluster transfer.
Figure 4.
Cluster transfer can occur in the presence of the biological reducing agent glutathione.
It is essential to know whether cluster transfer can occur in the presence of the biological reducing agent glutathione. Therefore cluster transfer assays were performed in vitro at 37°C in the presence of 5 mM glutathione (GSH) for the specified length of time and then analyzed by Native PAGE. Fd with its [2Fe-2S] cluster (holo-Fd) was run as a reference. The upper gel is not stained and shows the transfer of the [2Fe-2S] cluster by the visible red bands on the gel. The lower panel is the same gel after staining with Coomassie.
Figure 5.
Transfer of labile iron from NAF-1 to mitochondria.
Upper panel. Pseudo-colored images of permeabilized h9c2 cells labeled with red rhodamine B-[(1,10-phenanthrolin-5-yl)] benzyl ester (RPA) to trace iron in the mitochondrial matrix and then measured for fluorescence every two minutes. NAF-1 (WT or H114C mutated) was added to either 0, 5, 10, or 20 μM concentrations after 4 minutes (only 20 μM profiles are shown). The pseudocolor of the cells indicates the relative levels of mitochondrial RPA fluorescence (orange: high; blue: low). The liposoluble permeant FHQ, was added to 5 μM after 22 min in order to attain maximum quenching. Lower. Plot of RPA fluorescence is given in terms of arbitrary units (a.u.) obtained by analyzing individual cell fluorescence with Image J as described in Experimental Procedures. The RPA fluorescence is the average of four independent runs.
Figure 6.
Effect of pioglitazone and resveratrol on NAF-1: cluster stabilization and abrogation of iron transfer to mitochondria.
Upper panel. Kinetics of NAF-1 stability in the absence and presence of pioglitazone monitored spectrophotometrically (458 nm) at 37°C at pH 7.0 (NAF-1 and pioglitazone 20 µM each). The half-decay time of the absorbance corresponding to NAF-1 (2Fe-2S) cluster was raised by pioglitazone from t1/2 = 1000±160 min (filled circles) to t1/2 = 4700±350 min (open circles). Likewise, 20 µM resveratrol delayed NAF-1 cluster decomposition with a t1/2 = 6800±500 min (open circles). Lower. Effect of drugs on NAF-1 ability to transfer labile iron to RPA labeled mitochondria using permeabilized h9c2 cells. Data acquisition, analysis from fluorescence images and plotting were done as described in the legend to Fig. 5. Addition of NAF-1 (10 μM) to RPA-labeled permeabilized cells at 4 min generated a fast quenching of RPA (red) whereas addition of none (blue) was steady until supplemented with the permeant FHQ (5 μM) at 22 min, which led to maximal attainable quenching. The addition of NAF-1 preincubated with resveratrol (20 μM) (purple) or pioglitazone (yellow) abrogated cluster transfer as evidenced by the absence of RPA fluorescent quenching, again, until the system was challenged with the permeant FHQ.