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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.

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Evidence for indirect heme transfer from human methemoglobin to apoShp. Methemoglobin (total 5.8 μM heme) was reacted with 28 μM apoShp or 40 μM H64Y/V68F apomyoglobin in 20 mM Tris-HCl, pH 8.0, and 0.45 M sucrose, and absorbance time courses at 425 and 405 or 410 nm were recorded. (A) The normalized spectral changes Δ(A425-A405) (green curve) and Δ(A425-A410) (red curve) for the methemoglobin/apoShp and methemoglobin/apomyoglobin reactions, respectively. The black curves are the theoretical curves obtained by fitting the data to a two exponential equation. (B) Δ(A425-A405) of the methemoglobin/apoShp reaction does not fit a single exponential equation. The dashed and solid curves are respectively the observed data and theoretical curve from fitting of the data to a single exponential equation.
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Figure 1: Evidence for indirect heme transfer from human methemoglobin to apoShp. Methemoglobin (total 5.8 μM heme) was reacted with 28 μM apoShp or 40 μM H64Y/V68F apomyoglobin in 20 mM Tris-HCl, pH 8.0, and 0.45 M sucrose, and absorbance time courses at 425 and 405 or 410 nm were recorded. (A) The normalized spectral changes Δ(A425-A405) (green curve) and Δ(A425-A410) (red curve) for the methemoglobin/apoShp and methemoglobin/apomyoglobin reactions, respectively. The black curves are the theoretical curves obtained by fitting the data to a two exponential equation. (B) Δ(A425-A405) of the methemoglobin/apoShp reaction does not fit a single exponential equation. The dashed and solid curves are respectively the observed data and theoretical curve from fitting of the data to a single exponential equation.

Mentions: Heme-binding form of Shp (holoShp) was formed in the incubation of apoShp with human methemoglobin [18]. It was not known whether the formation of holoShp was due to direct heme transfer from methemoglobin or indirect scavenge of ferric heme dissociated from methemoglobin. To examine this issue, the rate for formation of holoShp was compared with that of heme dissociation from methemoglobin. Limited methemoglobin (total 5.8 μM heme) was incubated with 7 to 56 μM apoShp at 25°C, and the formation of holoShp was monitored by absorbance change at 405 and 425 nm, which present the loss and gain of heme by methemoglobin and apoShp, respectively. The time courses of normalized absorbance change, Δ(A425-A405), fit a two exponential equation (Fig. 1A), but not a single exponential equation (Fig. 1B). Fitting of the data to a two exponential equation resulted in two first-order rate constants. The values of the rate constants did not change significantly with [apoShp] from 7 to 56 μM, giving the mean values ± standard deviation of 0.0065 ± 0.0013 and 0.00041 ± 0.00012 s-1 for the rate constants of the fast and slow phases, respectively. The biphasic kinetics and rate constants are similar to those in the dissociation of heme from methemoglobin using H64Y/V68F apomyoglobin as a heme scavenger [22], in which the fast and slow phases are heme dissociation from the β and α subunits, respectively [22,23]. To confirm these similarities, the dissociation of heme from methemoglobin using H64Y/V68F apomyoglobin was performed under the same conditions. Δ(A425-A410) associated with the loss and gain of heme by methemoglobin and apomyoglobin, respectively, was kinetically biphasic with 0.0058 ± 0.0002 and 0.00049 ± 0.00001 s-1 for the rate constants of the fast and slow phases, respectively (Fig. 1A). These rate constants are close to those in the methemoglobin/apoShp reaction. These results suggest that Shp does not directly acquire heme from human methemoglobin.


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)

Evidence for indirect heme transfer from human methemoglobin to apoShp. Methemoglobin (total 5.8 μM heme) was reacted with 28 μM apoShp or 40 μM H64Y/V68F apomyoglobin in 20 mM Tris-HCl, pH 8.0, and 0.45 M sucrose, and absorbance time courses at 425 and 405 or 410 nm were recorded. (A) The normalized spectral changes Δ(A425-A405) (green curve) and Δ(A425-A410) (red curve) for the methemoglobin/apoShp and methemoglobin/apomyoglobin reactions, respectively. The black curves are the theoretical curves obtained by fitting the data to a two exponential equation. (B) Δ(A425-A405) of the methemoglobin/apoShp reaction does not fit a single exponential equation. The dashed and solid curves are respectively the observed data and theoretical curve from fitting of the data to a single exponential equation.
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Related In: Results  -  Collection

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Figure 1: Evidence for indirect heme transfer from human methemoglobin to apoShp. Methemoglobin (total 5.8 μM heme) was reacted with 28 μM apoShp or 40 μM H64Y/V68F apomyoglobin in 20 mM Tris-HCl, pH 8.0, and 0.45 M sucrose, and absorbance time courses at 425 and 405 or 410 nm were recorded. (A) The normalized spectral changes Δ(A425-A405) (green curve) and Δ(A425-A410) (red curve) for the methemoglobin/apoShp and methemoglobin/apomyoglobin reactions, respectively. The black curves are the theoretical curves obtained by fitting the data to a two exponential equation. (B) Δ(A425-A405) of the methemoglobin/apoShp reaction does not fit a single exponential equation. The dashed and solid curves are respectively the observed data and theoretical curve from fitting of the data to a single exponential equation.
Mentions: Heme-binding form of Shp (holoShp) was formed in the incubation of apoShp with human methemoglobin [18]. It was not known whether the formation of holoShp was due to direct heme transfer from methemoglobin or indirect scavenge of ferric heme dissociated from methemoglobin. To examine this issue, the rate for formation of holoShp was compared with that of heme dissociation from methemoglobin. Limited methemoglobin (total 5.8 μM heme) was incubated with 7 to 56 μM apoShp at 25°C, and the formation of holoShp was monitored by absorbance change at 405 and 425 nm, which present the loss and gain of heme by methemoglobin and apoShp, respectively. The time courses of normalized absorbance change, Δ(A425-A405), fit a two exponential equation (Fig. 1A), but not a single exponential equation (Fig. 1B). Fitting of the data to a two exponential equation resulted in two first-order rate constants. The values of the rate constants did not change significantly with [apoShp] from 7 to 56 μM, giving the mean values ± standard deviation of 0.0065 ± 0.0013 and 0.00041 ± 0.00012 s-1 for the rate constants of the fast and slow phases, respectively. The biphasic kinetics and rate constants are similar to those in the dissociation of heme from methemoglobin using H64Y/V68F apomyoglobin as a heme scavenger [22], in which the fast and slow phases are heme dissociation from the β and α subunits, respectively [22,23]. To confirm these similarities, the dissociation of heme from methemoglobin using H64Y/V68F apomyoglobin was performed under the same conditions. Δ(A425-A410) associated with the loss and gain of heme by methemoglobin and apomyoglobin, respectively, was kinetically biphasic with 0.0058 ± 0.0002 and 0.00049 ± 0.00001 s-1 for the rate constants of the fast and slow phases, respectively (Fig. 1A). These rate constants are close to those in the methemoglobin/apoShp reaction. These results suggest that Shp does not directly acquire heme from human methemoglobin.

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