Limits...
The surface protein Shr of Streptococcus pyogenes binds heme and transfers it to the streptococcal heme-binding protein Shp.

Zhu H, Liu M, Lei B - BMC Microbiol. (2008)

Bottom Line: These results suggest that Shr directly transfers its heme to Shp.In addition, the rates of heme transfer from human hemoglobin to apoShp are close to those of simple ferric heme dissociation from hemoglobin, suggesting that methemoglobin does not directly transfer its heme to apoShp.These results suggest the possibility that Shr is a source of heme for Shp and that the Shr-to-Shp heme transfer is a step of the heme acquisition process in S. pyogenes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59717, USA. dzhuhui@yahoo.com.cn

ABSTRACT

Background: The heme acquisition machinery in Streptococcus pyogenes is believed to consist of the surface proteins, Shr and Shp, and heme-specific ATP-binding cassette transporter HtsABC. Shp has been shown to rapidly transfer its heme to the lipoprotein component, HtsA, of HtsABC. The function of Shr and the heme source of Shp have not been established.

Results: The objective of this study was to determine whether Shr binds heme and is a heme source of Shp. To achieve the objective, recombinant Shr protein was prepared. The purified Shr displays a spectrum typical of hemoproteins, indicating that Shr binds heme and acquires heme from Escherichia coli hemoproteins in vivo. Spectral analysis of Shr and Shp isolated from a mixture of Shr and heme-free Shp (apoShp) indicates that Shr and apoShp lost and gained heme, respectively; whereas Shr did not efficiently lose its heme in incubation with apoHtsA under the identical conditions. These results suggest that Shr directly transfers its heme to Shp. In addition, the rates of heme transfer from human hemoglobin to apoShp are close to those of simple ferric heme dissociation from hemoglobin, suggesting that methemoglobin does not directly transfer its heme to apoShp.

Conclusion: We have demonstrated that recombinant Shr can acquire heme from E. coli hemoproteins in vivo and appears to directly transfer its heme to Shp and that Shp appears not to directly acquire heme from human methemoglobin. These results suggest the possibility that Shr is a source of heme for Shp and that the Shr-to-Shp heme transfer is a step of the heme acquisition process in S. pyogenes. Further characterization of the Shr/Shp/HtsA system would advance our understanding of the mechanism of heme acquisition in S. pyogenes.

Show MeSH

Related in: MedlinePlus

Spectral evidence for that Shr binds heme. Presented are the spectra of 15 μM Shr isolated (black solid curve), treated with 50 μM ferricyanide (red curve), and reduced by excess dithionite (dashed line) in 20 mM Tris-HCl, pH 8.0.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2266757&req=5

Figure 4: Spectral evidence for that Shr binds heme. Presented are the spectra of 15 μM Shr isolated (black solid curve), treated with 50 μM ferricyanide (red curve), and reduced by excess dithionite (dashed line) in 20 mM Tris-HCl, pH 8.0.

Mentions: The pellet of E. coli expressing Shr was in light red color. Purified Shr was yellow and red at diluted and concentrated concentrations, respectively, while the proteins obtained from the control E. coli from the SP Sepharose chromatography did not have absorption in the visible region, indicating that Shr has an associated chromophore. In the presence of excess dithionite, the protein displays the absorption peaks at 426, 528, and 560 nm (Fig. 4), a spectrum typical of ferrous heme in a hexacoordinate form, indicating that the chromophore is heme. Shr as isolated had a broad Soret peak and the small resolved α band at 560 nm, suggesting that the protein was a mixture of ferric and ferrous heme complexes. To confirm this, the protein was incubated with ferricyanide, and excess ferricyanide was removed from the protein by a G-25 Sepharose column. The Soret peak of the ferricyaide-treated Shr was indeed shifted to that of ferric heme complex (Fig. 3), indicating that Shr as isolated was a mixture of ferric and ferrous heme complexes. The results of the pyridine hemochrome assay further confirm that the chromophore is heme. The spectrum of the chromophore derived from Shr in this assay was identical to the one of pyridine hemochrome derived from an authentic hemoprotein (data not shown). The molar ratio of associated heme to Shr was 0.8 after adjustment according to the estimated purity of the purified Shr. These results indicate that the chromophore associated with Shr is heme and that Shr can acquire heme from E. coli hemoproteins in vivo.


The surface protein Shr of Streptococcus pyogenes binds heme and transfers it to the streptococcal heme-binding protein Shp.

Zhu H, Liu M, Lei B - BMC Microbiol. (2008)

Spectral evidence for that Shr binds heme. Presented are the spectra of 15 μM Shr isolated (black solid curve), treated with 50 μM ferricyanide (red curve), and reduced by excess dithionite (dashed line) in 20 mM Tris-HCl, pH 8.0.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2266757&req=5

Figure 4: Spectral evidence for that Shr binds heme. Presented are the spectra of 15 μM Shr isolated (black solid curve), treated with 50 μM ferricyanide (red curve), and reduced by excess dithionite (dashed line) in 20 mM Tris-HCl, pH 8.0.
Mentions: The pellet of E. coli expressing Shr was in light red color. Purified Shr was yellow and red at diluted and concentrated concentrations, respectively, while the proteins obtained from the control E. coli from the SP Sepharose chromatography did not have absorption in the visible region, indicating that Shr has an associated chromophore. In the presence of excess dithionite, the protein displays the absorption peaks at 426, 528, and 560 nm (Fig. 4), a spectrum typical of ferrous heme in a hexacoordinate form, indicating that the chromophore is heme. Shr as isolated had a broad Soret peak and the small resolved α band at 560 nm, suggesting that the protein was a mixture of ferric and ferrous heme complexes. To confirm this, the protein was incubated with ferricyanide, and excess ferricyanide was removed from the protein by a G-25 Sepharose column. The Soret peak of the ferricyaide-treated Shr was indeed shifted to that of ferric heme complex (Fig. 3), indicating that Shr as isolated was a mixture of ferric and ferrous heme complexes. The results of the pyridine hemochrome assay further confirm that the chromophore is heme. The spectrum of the chromophore derived from Shr in this assay was identical to the one of pyridine hemochrome derived from an authentic hemoprotein (data not shown). The molar ratio of associated heme to Shr was 0.8 after adjustment according to the estimated purity of the purified Shr. These results indicate that the chromophore associated with Shr is heme and that Shr can acquire heme from E. coli hemoproteins in vivo.

Bottom Line: These results suggest that Shr directly transfers its heme to Shp.In addition, the rates of heme transfer from human hemoglobin to apoShp are close to those of simple ferric heme dissociation from hemoglobin, suggesting that methemoglobin does not directly transfer its heme to apoShp.These results suggest the possibility that Shr is a source of heme for Shp and that the Shr-to-Shp heme transfer is a step of the heme acquisition process in S. pyogenes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59717, USA. dzhuhui@yahoo.com.cn

ABSTRACT

Background: The heme acquisition machinery in Streptococcus pyogenes is believed to consist of the surface proteins, Shr and Shp, and heme-specific ATP-binding cassette transporter HtsABC. Shp has been shown to rapidly transfer its heme to the lipoprotein component, HtsA, of HtsABC. The function of Shr and the heme source of Shp have not been established.

Results: The objective of this study was to determine whether Shr binds heme and is a heme source of Shp. To achieve the objective, recombinant Shr protein was prepared. The purified Shr displays a spectrum typical of hemoproteins, indicating that Shr binds heme and acquires heme from Escherichia coli hemoproteins in vivo. Spectral analysis of Shr and Shp isolated from a mixture of Shr and heme-free Shp (apoShp) indicates that Shr and apoShp lost and gained heme, respectively; whereas Shr did not efficiently lose its heme in incubation with apoHtsA under the identical conditions. These results suggest that Shr directly transfers its heme to Shp. In addition, the rates of heme transfer from human hemoglobin to apoShp are close to those of simple ferric heme dissociation from hemoglobin, suggesting that methemoglobin does not directly transfer its heme to apoShp.

Conclusion: We have demonstrated that recombinant Shr can acquire heme from E. coli hemoproteins in vivo and appears to directly transfer its heme to Shp and that Shp appears not to directly acquire heme from human methemoglobin. These results suggest the possibility that Shr is a source of heme for Shp and that the Shr-to-Shp heme transfer is a step of the heme acquisition process in S. pyogenes. Further characterization of the Shr/Shp/HtsA system would advance our understanding of the mechanism of heme acquisition in S. pyogenes.

Show MeSH
Related in: MedlinePlus