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The role of CyaY in iron sulfur cluster assembly on the E. coli IscU scaffold protein.

Iannuzzi C, Adinolfi S, Howes BD, Garcia-Serres R, Clémancey M, Latour JM, Smulevich G, Pastore A - PLoS ONE (2011)

Bottom Line: Similar studies on the eukaryotic proteins have however suggested for frataxin a role as an activator.Our studies independently confirm that CyaY slows down the reaction and shed new light onto the mechanism by which CyaY works.We observe that the presence of CyaY does not alter the relative ratio between [2Fe2S](2+) and [4Fe4S](2+) but directly affects enzymatic activity.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council National Institute for Medical Research, London, United Kingdom.

ABSTRACT
Progress in understanding the mechanism underlying the enzymatic formation of iron-sulfur clusters is difficult since it involves a complex reaction and a multi-component system. By exploiting different spectroscopies, we characterize the effect on the enzymatic kinetics of cluster formation of CyaY, the bacterial ortholog of frataxin, on cluster formation on the scaffold protein IscU. Frataxin/CyaY is a highly conserved protein implicated in an incurable ataxia in humans. Previous studies had suggested a role of CyaY as an inhibitor of iron sulfur cluster formation. Similar studies on the eukaryotic proteins have however suggested for frataxin a role as an activator. Our studies independently confirm that CyaY slows down the reaction and shed new light onto the mechanism by which CyaY works. We observe that the presence of CyaY does not alter the relative ratio between [2Fe2S](2+) and [4Fe4S](2+) but directly affects enzymatic activity.

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Related in: MedlinePlus

Time dependence of FeS cluster formation deduced from the Mössbauer experiments.The individual contributions of the different complexes are estimated at each time point (5, 15 and 30 min) in the absence (left) and in the presence (right) of CyaY.
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pone-0021992-g004: Time dependence of FeS cluster formation deduced from the Mössbauer experiments.The individual contributions of the different complexes are estimated at each time point (5, 15 and 30 min) in the absence (left) and in the presence (right) of CyaY.

Mentions: The time dependence of Fe-S cluster formation was followed by Mössbauer spectroscopy upon freezing 57Fe-enriched samples at different time points after initiation of the reaction. Three separate time course experiments were monitored with duration times in the range 30 to 120 min. A representative experiment recorded over a 0–30 min period in the absence (Figures 2A–D) and in the presence (Figures 2E–H) of CyaY is presented. In the absence of CyaY, the initial spectrum (Figure 2A) consists of a broad asymmetric doublet that is associated with FeII species, mostly tetrahedral ferrous thiolates [18], [25]. The spectrum recorded after 5 min (Figure 2B) shows the appearance of a weak doublet in the velocity region −0.5–1 mm/s. As time elapses (Figures 2C,D), the left absorption corresponding to the low energy peak of this doublet increases but its right counterpart both grows in intensity and undergoes some deformation owing to the development of a new absorption at ca. 1 mm/s. This behaviour is consistent with the successive appearance of at least two species. The spectra in Figures 2B–D were simulated as mixtures of the ferrous components present in Figure 2A and the [2Fe-2S]2+ and [4Fe-4S]2+ clusters, that could be assigned according to the literature parameters cited above. In all cases excellent fits were obtained. The spectrum associated with the Fe-S clusters after subtraction of the ferrous component from the spectrum in Figure 2D and its deconvolution shows that (i) initially the [2Fe-2S]2+ cluster grows faster so that it dominates at 5 min (8% of total iron vs 1% for the [4Fe-4S]2+ clusters) and at 15 min (12% vs 8%) but (ii) then the [4Fe-4S]2+ grows faster so that at 30 min the two have the same weight (17% of total iron vs 17% for the [2Fe-2S]2+ clusters) (Figure 3). The time dependence of the formation of the clusters is illustrated in Figure 4.


The role of CyaY in iron sulfur cluster assembly on the E. coli IscU scaffold protein.

Iannuzzi C, Adinolfi S, Howes BD, Garcia-Serres R, Clémancey M, Latour JM, Smulevich G, Pastore A - PLoS ONE (2011)

Time dependence of FeS cluster formation deduced from the Mössbauer experiments.The individual contributions of the different complexes are estimated at each time point (5, 15 and 30 min) in the absence (left) and in the presence (right) of CyaY.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3140489&req=5

pone-0021992-g004: Time dependence of FeS cluster formation deduced from the Mössbauer experiments.The individual contributions of the different complexes are estimated at each time point (5, 15 and 30 min) in the absence (left) and in the presence (right) of CyaY.
Mentions: The time dependence of Fe-S cluster formation was followed by Mössbauer spectroscopy upon freezing 57Fe-enriched samples at different time points after initiation of the reaction. Three separate time course experiments were monitored with duration times in the range 30 to 120 min. A representative experiment recorded over a 0–30 min period in the absence (Figures 2A–D) and in the presence (Figures 2E–H) of CyaY is presented. In the absence of CyaY, the initial spectrum (Figure 2A) consists of a broad asymmetric doublet that is associated with FeII species, mostly tetrahedral ferrous thiolates [18], [25]. The spectrum recorded after 5 min (Figure 2B) shows the appearance of a weak doublet in the velocity region −0.5–1 mm/s. As time elapses (Figures 2C,D), the left absorption corresponding to the low energy peak of this doublet increases but its right counterpart both grows in intensity and undergoes some deformation owing to the development of a new absorption at ca. 1 mm/s. This behaviour is consistent with the successive appearance of at least two species. The spectra in Figures 2B–D were simulated as mixtures of the ferrous components present in Figure 2A and the [2Fe-2S]2+ and [4Fe-4S]2+ clusters, that could be assigned according to the literature parameters cited above. In all cases excellent fits were obtained. The spectrum associated with the Fe-S clusters after subtraction of the ferrous component from the spectrum in Figure 2D and its deconvolution shows that (i) initially the [2Fe-2S]2+ cluster grows faster so that it dominates at 5 min (8% of total iron vs 1% for the [4Fe-4S]2+ clusters) and at 15 min (12% vs 8%) but (ii) then the [4Fe-4S]2+ grows faster so that at 30 min the two have the same weight (17% of total iron vs 17% for the [2Fe-2S]2+ clusters) (Figure 3). The time dependence of the formation of the clusters is illustrated in Figure 4.

Bottom Line: Similar studies on the eukaryotic proteins have however suggested for frataxin a role as an activator.Our studies independently confirm that CyaY slows down the reaction and shed new light onto the mechanism by which CyaY works.We observe that the presence of CyaY does not alter the relative ratio between [2Fe2S](2+) and [4Fe4S](2+) but directly affects enzymatic activity.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council National Institute for Medical Research, London, United Kingdom.

ABSTRACT
Progress in understanding the mechanism underlying the enzymatic formation of iron-sulfur clusters is difficult since it involves a complex reaction and a multi-component system. By exploiting different spectroscopies, we characterize the effect on the enzymatic kinetics of cluster formation of CyaY, the bacterial ortholog of frataxin, on cluster formation on the scaffold protein IscU. Frataxin/CyaY is a highly conserved protein implicated in an incurable ataxia in humans. Previous studies had suggested a role of CyaY as an inhibitor of iron sulfur cluster formation. Similar studies on the eukaryotic proteins have however suggested for frataxin a role as an activator. Our studies independently confirm that CyaY slows down the reaction and shed new light onto the mechanism by which CyaY works. We observe that the presence of CyaY does not alter the relative ratio between [2Fe2S](2+) and [4Fe4S](2+) but directly affects enzymatic activity.

Show MeSH
Related in: MedlinePlus