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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

RR and electronic absorption spectra of Fe-S clusters assembled on E. coli IscU.(A) Electronic absorption spectra in both normal and second derivative presentations of [2Fe-2S]2+ and [4Fe-4S]2+ clusters, and (B) RR spectra of almost pure [2Fe-2S]2+ and [4Fe-4S]2+ clusters. RR experimental conditions: excitation wavelength 413.1 nm, spectral resolution 1 cm−1, laser power at the sample 55 mW; ([2Fe-2S]2+) average of eleven spectra with 15 min integration time; ([4Fe-4S]2+) average of nine spectra with 10 min integration time. The asterisks indicate laser plasma lines that have been removed.
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pone-0021992-g001: RR and electronic absorption spectra of Fe-S clusters assembled on E. coli IscU.(A) Electronic absorption spectra in both normal and second derivative presentations of [2Fe-2S]2+ and [4Fe-4S]2+ clusters, and (B) RR spectra of almost pure [2Fe-2S]2+ and [4Fe-4S]2+ clusters. RR experimental conditions: excitation wavelength 413.1 nm, spectral resolution 1 cm−1, laser power at the sample 55 mW; ([2Fe-2S]2+) average of eleven spectra with 15 min integration time; ([4Fe-4S]2+) average of nine spectra with 10 min integration time. The asterisks indicate laser plasma lines that have been removed.

Mentions: The evolution of Fe-S cluster formation has extensively been described using electronic absorption spectroscopy [11], [21]. Electronic absorption spectra of Fe-S proteins provide clear evidence for the presence of Fe-S clusters allowing discrimination between [2Fe-2S]2+ and [4Fe-4S]2+ species. Similar to other Fe-S proteins, the 300–800 nm spectrum of the [2Fe-2S]2+ cluster formed on IscU is characterised by bands at 320, 405, and 455 nm and a shoulder at about 515 nm, as highlighted by the second derivative (D2) spectrum (Figure 1A, bottom). As the reaction proceeds, or upon reduction, the absorbance spectrum changes and the formation of the [4Fe-4S]2+ cluster can be readily identified on the basis of the single band detected at 415 nm (Figure 1A, top) [11], [18]. It should however be noted that the band at 415–420 nm overlaps the absorption bands of other iron complexes; we have for instance shown before that iron-loaded IscS and CyaY have bands around the same wavelength in the absence of any other component [13].


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)

RR and electronic absorption spectra of Fe-S clusters assembled on E. coli IscU.(A) Electronic absorption spectra in both normal and second derivative presentations of [2Fe-2S]2+ and [4Fe-4S]2+ clusters, and (B) RR spectra of almost pure [2Fe-2S]2+ and [4Fe-4S]2+ clusters. RR experimental conditions: excitation wavelength 413.1 nm, spectral resolution 1 cm−1, laser power at the sample 55 mW; ([2Fe-2S]2+) average of eleven spectra with 15 min integration time; ([4Fe-4S]2+) average of nine spectra with 10 min integration time. The asterisks indicate laser plasma lines that have been removed.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0021992-g001: RR and electronic absorption spectra of Fe-S clusters assembled on E. coli IscU.(A) Electronic absorption spectra in both normal and second derivative presentations of [2Fe-2S]2+ and [4Fe-4S]2+ clusters, and (B) RR spectra of almost pure [2Fe-2S]2+ and [4Fe-4S]2+ clusters. RR experimental conditions: excitation wavelength 413.1 nm, spectral resolution 1 cm−1, laser power at the sample 55 mW; ([2Fe-2S]2+) average of eleven spectra with 15 min integration time; ([4Fe-4S]2+) average of nine spectra with 10 min integration time. The asterisks indicate laser plasma lines that have been removed.
Mentions: The evolution of Fe-S cluster formation has extensively been described using electronic absorption spectroscopy [11], [21]. Electronic absorption spectra of Fe-S proteins provide clear evidence for the presence of Fe-S clusters allowing discrimination between [2Fe-2S]2+ and [4Fe-4S]2+ species. Similar to other Fe-S proteins, the 300–800 nm spectrum of the [2Fe-2S]2+ cluster formed on IscU is characterised by bands at 320, 405, and 455 nm and a shoulder at about 515 nm, as highlighted by the second derivative (D2) spectrum (Figure 1A, bottom). As the reaction proceeds, or upon reduction, the absorbance spectrum changes and the formation of the [4Fe-4S]2+ cluster can be readily identified on the basis of the single band detected at 415 nm (Figure 1A, top) [11], [18]. It should however be noted that the band at 415–420 nm overlaps the absorption bands of other iron complexes; we have for instance shown before that iron-loaded IscS and CyaY have bands around the same wavelength in the absence of any other component [13].

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