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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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Kinetics of cluster formation.(A) Electronic absorption spectra obtained during the time course of Fe-S synthesis on E. coli IscU at the times indicated. (B) (a) Superposition of the RR spectra obtained during the time course of Fe-S synthesis in the time interval 30–60 min (no CyaY, black) and 30–90 min (+CyaY, red). (b) Superposition of the RR spectra for admixtures of the pure species shown in Figure 1: 50% [4Fe-4S]2+ /50% [2Fe-2S]2+ (red) and 40% [4Fe-4S]2+ /60% [2Fe-2S]2+ (black). RR experimental conditions: excitation wavelength 413.1 nm, spectral resolution 1 cm−1, laser power at the sample 50 mW; (no CyaY) average of 5 spectra with 10 min integration time; (+CyaY) average of 10 spectra with 10 min integration time. The spectra are normalised on the 294 cm−1 band of the [2Fe-2S]2+ cluster. The asterisk indicates a laser plasma line which has been removed.
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pone-0021992-g005: Kinetics of cluster formation.(A) Electronic absorption spectra obtained during the time course of Fe-S synthesis on E. coli IscU at the times indicated. (B) (a) Superposition of the RR spectra obtained during the time course of Fe-S synthesis in the time interval 30–60 min (no CyaY, black) and 30–90 min (+CyaY, red). (b) Superposition of the RR spectra for admixtures of the pure species shown in Figure 1: 50% [4Fe-4S]2+ /50% [2Fe-2S]2+ (red) and 40% [4Fe-4S]2+ /60% [2Fe-2S]2+ (black). RR experimental conditions: excitation wavelength 413.1 nm, spectral resolution 1 cm−1, laser power at the sample 50 mW; (no CyaY) average of 5 spectra with 10 min integration time; (+CyaY) average of 10 spectra with 10 min integration time. The spectra are normalised on the 294 cm−1 band of the [2Fe-2S]2+ cluster. The asterisk indicates a laser plasma line which has been removed.

Mentions: We then exploited the electronic absorption and RR spectral properties of the nearly pure forms of the [2Fe-2S]2+ and [4Fe-4S]2+ clusters assembled on IscU described above (Figure 1) to follow in parallel by both techniques the kinetics of Fe-S clusters formation in the absence and in the presence of CyaY at room temperature. The electronic absorption spectra observed at corresponding times in the absence and in the presence of CyaY are very similar (Figure 5A). In agreement with the Mössbauer spectra, in both sets of spectra, the variations indicate a progressive increase in the proportion of the [4Fe-4S]2+ clusters at the expense of [2Fe-2S]2+. In the presence of CyaY, the time course of Fe-S formation is extended, indicating that the rate of Fe-S synthesis is reduced. In both cases, the variations observed in the electronic absorption spectra suggest that the conversion of [2Fe-2S]2+ to [4Fe-4S]2+ is already at an advanced stage after ca. 25 min, coincident with the beginning of the RR experiment. However, the RR spectra could not be analysed prior to 30 min because of their poor signal-to-noise ratio.


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)

Kinetics of cluster formation.(A) Electronic absorption spectra obtained during the time course of Fe-S synthesis on E. coli IscU at the times indicated. (B) (a) Superposition of the RR spectra obtained during the time course of Fe-S synthesis in the time interval 30–60 min (no CyaY, black) and 30–90 min (+CyaY, red). (b) Superposition of the RR spectra for admixtures of the pure species shown in Figure 1: 50% [4Fe-4S]2+ /50% [2Fe-2S]2+ (red) and 40% [4Fe-4S]2+ /60% [2Fe-2S]2+ (black). RR experimental conditions: excitation wavelength 413.1 nm, spectral resolution 1 cm−1, laser power at the sample 50 mW; (no CyaY) average of 5 spectra with 10 min integration time; (+CyaY) average of 10 spectra with 10 min integration time. The spectra are normalised on the 294 cm−1 band of the [2Fe-2S]2+ cluster. The asterisk indicates a laser plasma line which has been removed.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3140489&req=5

pone-0021992-g005: Kinetics of cluster formation.(A) Electronic absorption spectra obtained during the time course of Fe-S synthesis on E. coli IscU at the times indicated. (B) (a) Superposition of the RR spectra obtained during the time course of Fe-S synthesis in the time interval 30–60 min (no CyaY, black) and 30–90 min (+CyaY, red). (b) Superposition of the RR spectra for admixtures of the pure species shown in Figure 1: 50% [4Fe-4S]2+ /50% [2Fe-2S]2+ (red) and 40% [4Fe-4S]2+ /60% [2Fe-2S]2+ (black). RR experimental conditions: excitation wavelength 413.1 nm, spectral resolution 1 cm−1, laser power at the sample 50 mW; (no CyaY) average of 5 spectra with 10 min integration time; (+CyaY) average of 10 spectra with 10 min integration time. The spectra are normalised on the 294 cm−1 band of the [2Fe-2S]2+ cluster. The asterisk indicates a laser plasma line which has been removed.
Mentions: We then exploited the electronic absorption and RR spectral properties of the nearly pure forms of the [2Fe-2S]2+ and [4Fe-4S]2+ clusters assembled on IscU described above (Figure 1) to follow in parallel by both techniques the kinetics of Fe-S clusters formation in the absence and in the presence of CyaY at room temperature. The electronic absorption spectra observed at corresponding times in the absence and in the presence of CyaY are very similar (Figure 5A). In agreement with the Mössbauer spectra, in both sets of spectra, the variations indicate a progressive increase in the proportion of the [4Fe-4S]2+ clusters at the expense of [2Fe-2S]2+. In the presence of CyaY, the time course of Fe-S formation is extended, indicating that the rate of Fe-S synthesis is reduced. In both cases, the variations observed in the electronic absorption spectra suggest that the conversion of [2Fe-2S]2+ to [4Fe-4S]2+ is already at an advanced stage after ca. 25 min, coincident with the beginning of the RR experiment. However, the RR spectra could not be analysed prior to 30 min because of their poor signal-to-noise ratio.

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