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The 2-Cys Peroxiredoxin Alkyl Hydroperoxide Reductase C Binds Heme and Participates in Its Intracellular Availability in Streptococcus agalactiae

Lechardeur, Delphine, Fernandez, Annabelle, Robert, Bruno, Gaudu, Philippe, Trieu-Cuot, Patrick, Lamberet, Gilles, Gruss, Alexandra·2010·Journal of Biological Chemistry
DOI 10.1074/jbc.m109.024505
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Abstract

Heme is a redox-reactive molecule with vital and complex roles in bacterial metabolism, survival, and virulence. However, few intracellular heme partners were identified to date and are not well conserved in bacteria. The opportunistic pathogen Streptococcus agalactiae (group B Streptococcus) is a heme auxotroph, which acquires exogenous heme to activate an aerobic respiratory chain. We identified the alkyl hydroperoxide reductase AhpC, a member of the highly conserved thiol-dependent 2-Cys peroxiredoxins, as a heme-binding protein. AhpC binds hemin with a K(d) of 0.5 microm and a 1:1 stoichiometry. Mutagenesis of cysteines revealed that hemin binding is dissociable from catalytic activity and multimerization. AhpC reductase activity was unchanged upon interaction with heme in vitro and in vivo. A group B Streptococcus ahpC mutant displayed attenuation of two heme-dependent functions, respiration and activity of a heterologous catalase, suggesting a role for AhpC in heme intracellular fate. In support of this hypothesis, AhpC-bound hemin was protected from chemical degradation in vitro. Our results reveal for the first time a role for AhpC as a heme-binding protein.

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Figures

FIGURE 1 AhpC is a hemin-binding protein. A, purified His-AhpC binds to hemin-conjugated agarose. HisAhpC (50 𝛍 M) was incubated with the indicated amount of hemin in 50 mM Tris-HCl, pH 7.4, containing 50 mM NaCl for 15 min prior to hemin-agarose addition. After washings, bound protein was eluted from the resin with 2 × Laemmli sample buffer and processed for SDS-PAGE, followed by Coomassie Blue staining. Binding to hemin-agarose was inhibited by increasing hemin concentrations. B, UV-visible absorption spectrum of the AhpC-hemin complex. Purified holo-AhpC exhibits a Soret band at 414 nm and 𝛃 and 𝛂 bands at 535 and 566 nm,respectively( inset ). C, resonanceRamanspectrumofAhpC-hemin.Highfrequencyregionoftheresonance spectrumofthecomplexatpH7.4wasobtainedwith406.7nmlaserexcitation.The1632and1638cm -1 bands comprising 𝛎 10 were obtained by curve fitting. D, AhpC and hemin co-migrate on SDS-PAGE. His-AhpC alone ( left panel, left lane ) or in complex with hemin ( left panel, right lane ) were separated by SDS-PAGE without dithiothreitol as a mixture of disulfide-linked dimers (46 kDa) and monomers (23 kDa) ( left panel, upper and lower bands , respectively). The gel was stained for hemin-associated peroxidase activity with tetramethylbenzidine ( TMBZ , right panel ), showing that AhpC monomers and dimers both interact with hemin. No tetramethylbenzidine staining was observed with AhpC alone ( right panel, left lane ).
FIGURE 1 AhpC is a hemin-binding protein. A, purified His-AhpC binds to hemin-conjugated agarose. HisAhpC (50 𝛍 M) was incubated with the indicated amount of hemin in 50 mM Tris-HCl, pH 7.4, containing 50 mM NaCl for 15 min prior to hemin-agarose addition. After washings, bound protein was eluted from the resin with 2 × Laemmli sample buffer and processed for SDS-PAGE, followed by Coomassie Blue staining. Binding to hemin-agarose was inhibited by increasing hemin concentrations. B, UV-visible absorption spectrum of the AhpC-hemin complex. Purified holo-AhpC exhibits a Soret band at 414 nm and 𝛃 and 𝛂 bands at 535 and 566 nm,respectively( inset ). C, resonanceRamanspectrumofAhpC-hemin.Highfrequencyregionoftheresonance spectrumofthecomplexatpH7.4wasobtainedwith406.7nmlaserexcitation.The1632and1638cm -1 bands comprising 𝛎 10 were obtained by curve fitting. D, AhpC and hemin co-migrate on SDS-PAGE. His-AhpC alone ( left panel, left lane ) or in complex with hemin ( left panel, right lane ) were separated by SDS-PAGE without dithiothreitol as a mixture of disulfide-linked dimers (46 kDa) and monomers (23 kDa) ( left panel, upper and lower bands , respectively). The gel was stained for hemin-associated peroxidase activity with tetramethylbenzidine ( TMBZ , right panel ), showing that AhpC monomers and dimers both interact with hemin. No tetramethylbenzidine staining was observed with AhpC alone ( right panel, left lane ).CC-BY · From the PDF
FIGURE 2 Titration curves of AhpC WT and cysteine mutated with hemin. WT ( A ) and cysteine-mutated ( B ) AhpC (5 𝛍 M) were titrated with increasing increments of hemin. A 414 was plotted against hemin concentration. Curves are representative of three independent experiments and were fitted using the nonlinear regression function of GraphPad Prism 4 software with the equation defined under 'Experimental Procedures.' abs , absorbance.
FIGURE 2 Titration curves of AhpC WT and cysteine mutated with hemin. WT ( A ) and cysteine-mutated ( B ) AhpC (5 𝛍 M) were titrated with increasing increments of hemin. A 414 was plotted against hemin concentration. Curves are representative of three independent experiments and were fitted using the nonlinear regression function of GraphPad Prism 4 software with the equation defined under 'Experimental Procedures.' abs , absorbance.CC-BY · From the PDF
FIGURE 3 AhpC binds hemin in vivo . A, E. coli lysates expressing HA-AhpC were incubated with hemin-agarose. HA-AhpC was eluted from hemin-conjugated resin as seen with an anti-HA antibody. B, purified MBP-AhpC from ALA-treated E. coli exhibits a red color (as also observed for HA-AhpC under similar conditions; data not shown). E. coli strains carrying the pMAL-AhpC expression vector were grown to A 600 = 0.3 at 37 °C, cooled down to room temperature,andfurthergrownatroomtemperaturefor16hinthepresence of 0.25 mM isopropyl 1-thio𝛃 -D-galactopyranoside and 1 mM ALA, a heme precursor that stimulates its synthesis. Cells were lysed, and the MBP-AhpC protein was purified on amylose resin (see 'Experimental Procedures'). C, visible spectra of MBP-AhpC purified from E. coli treated with ALA or not as in B . Equivalent concentrations (60 𝛍 M) of MBP-AhpC as shown in B were analyzed byUV-visible spectrophotometry. Only the spectra corresponding to the protein isolated from ALA-treated cells exhibited a Soret band, further suggesting that heterologous overexpression of MBP-AhpC captured a fraction of overproducedhemin.TheMBPtaghasnoaffinityforhemin(datanotshown).
FIGURE 3 AhpC binds hemin in vivo . A, E. coli lysates expressing HA-AhpC were incubated with hemin-agarose. HA-AhpC was eluted from hemin-conjugated resin as seen with an anti-HA antibody. B, purified MBP-AhpC from ALA-treated E. coli exhibits a red color (as also observed for HA-AhpC under similar conditions; data not shown). E. coli strains carrying the pMAL-AhpC expression vector were grown to A 600 = 0.3 at 37 °C, cooled down to room temperature,andfurthergrownatroomtemperaturefor16hinthepresence of 0.25 mM isopropyl 1-thio𝛃 -D-galactopyranoside and 1 mM ALA, a heme precursor that stimulates its synthesis. Cells were lysed, and the MBP-AhpC protein was purified on amylose resin (see 'Experimental Procedures'). C, visible spectra of MBP-AhpC purified from E. coli treated with ALA or not as in B . Equivalent concentrations (60 𝛍 M) of MBP-AhpC as shown in B were analyzed byUV-visible spectrophotometry. Only the spectra corresponding to the protein isolated from ALA-treated cells exhibited a Soret band, further suggesting that heterologous overexpression of MBP-AhpC captured a fraction of overproducedhemin.TheMBPtaghasnoaffinityforhemin(datanotshown).CC-BY · From the PDF
FIGURE 4 GBS respiration metabolism is attenuated in an ahpC mutant. A, growth of WT and ahpC GBS in respiration-permissive conditions. WT and ahpC GBS strains were grown for 5-6 h in aerated or respiration-permissive (1 𝛍 Mheminand10 𝛍 MvitaminK2 ( vitK 2 )) or nonpermissive (1 𝛍 Mheminonly) conditions in M17 supplemented with 1% glucose. The growth increase under respiration conditions was less pronounced for the ahpC GBS mutant. Results represent the means ± S.D. from at least 10 independent experiments. Asterisk denotes statistically significant reduction of respiration in the ahpC mutant compared with the WT as determined by Student's t test ( p < 0.05). B, comparative oxygen consumption rates in WT and ahpC mutants grown as in Fig. 5 A . Oxygen consumption was measured on whole cells grown under aerobic fermentation and respiration conditions with a Clarktype electrode as described under 'Experimental Procedures.' Oxygen consumption, which is characteristic of respiration metabolism when oxygen is the terminal electron acceptor, was markedly reduced in the ahpC mutant, showingthatrespirationrateisdecreased.Dataarenormalizedto A 600 = 1for both cultures and are means ± S.D. from three experiments.
FIGURE 4 GBS respiration metabolism is attenuated in an ahpC mutant. A, growth of WT and ahpC GBS in respiration-permissive conditions. WT and ahpC GBS strains were grown for 5-6 h in aerated or respiration-permissive (1 𝛍 Mheminand10 𝛍 MvitaminK2 ( vitK 2 )) or nonpermissive (1 𝛍 Mheminonly) conditions in M17 supplemented with 1% glucose. The growth increase under respiration conditions was less pronounced for the ahpC GBS mutant. Results represent the means ± S.D. from at least 10 independent experiments. Asterisk denotes statistically significant reduction of respiration in the ahpC mutant compared with the WT as determined by Student's t test ( p < 0.05). B, comparative oxygen consumption rates in WT and ahpC mutants grown as in Fig. 5 A . Oxygen consumption was measured on whole cells grown under aerobic fermentation and respiration conditions with a Clarktype electrode as described under 'Experimental Procedures.' Oxygen consumption, which is characteristic of respiration metabolism when oxygen is the terminal electron acceptor, was markedly reduced in the ahpC mutant, showingthatrespirationrateisdecreased.Dataarenormalizedto A 600 = 1for both cultures and are means ± S.D. from three experiments.CC-BY · From the PDF
FIGURE 5 Levels of a heterologous heme catalase are reduced by ahpC inactivation. The E. faecalis catalase A (KatA) was expressed in GBS WT ( A ) and ahpC ( B ) strains transformed with pLUMB5 plasmid. Bacteria were incubated overnight with the indicated concentrations of hemin ( 𝛍 M). Catalase activity was assessed by measuring H 2 O2 consumption on whole cells with the xylenol orange assay (see 'Experimental Procedures'). The presented results are representative of three independent experiments. The GBS control (no KatA) has no endogenous catalase activity in the presence of hemin (data not shown). C, KatA expression in WT and ahpC GBS. Cells were grown overnight in the presence of the indicated hemin concentration ( 𝛍 M). Equivalent amounts of protein (50 𝛍 g) were separated on SDS-PAGE and probed with anti-KatA polyclonal antibody. The presented results are representative of three independent experiments.
FIGURE 5 Levels of a heterologous heme catalase are reduced by ahpC inactivation. The E. faecalis catalase A (KatA) was expressed in GBS WT ( A ) and ahpC ( B ) strains transformed with pLUMB5 plasmid. Bacteria were incubated overnight with the indicated concentrations of hemin ( 𝛍 M). Catalase activity was assessed by measuring H 2 O2 consumption on whole cells with the xylenol orange assay (see 'Experimental Procedures'). The presented results are representative of three independent experiments. The GBS control (no KatA) has no endogenous catalase activity in the presence of hemin (data not shown). C, KatA expression in WT and ahpC GBS. Cells were grown overnight in the presence of the indicated hemin concentration ( 𝛍 M). Equivalent amounts of protein (50 𝛍 g) were separated on SDS-PAGE and probed with anti-KatA polyclonal antibody. The presented results are representative of three independent experiments.CC-BY · From the PDF
FIGURE6 AhpC-boundheminisprotectedfromH2O2-mediateddegradation. 20 𝛍 M hemin alone or in complex with 20 𝛍 M His-AhpC or 20 𝛍 M BSA were diluted in 50 mM Tris-HCl buffer, pH 7.0. Absorption spectra between 300 and 500 nm were taken at the indicated times before ( t = 0) and at the indicated times and following the addition of 800 𝛍 M H2O2. The decrease in absorbance intensities of the Soret peak of free hemin (at A 385 ) or hemin boundtoAhpCorBSA(at A 414 ) were determined from the spectra. Intensities at the indicated time points were subtracted from the respective zero values and plotted. The presented curves are representative of three independent experiments.
FIGURE6 AhpC-boundheminisprotectedfromH2O2-mediateddegradation. 20 𝛍 M hemin alone or in complex with 20 𝛍 M His-AhpC or 20 𝛍 M BSA were diluted in 50 mM Tris-HCl buffer, pH 7.0. Absorption spectra between 300 and 500 nm were taken at the indicated times before ( t = 0) and at the indicated times and following the addition of 800 𝛍 M H2O2. The decrease in absorbance intensities of the Soret peak of free hemin (at A 385 ) or hemin boundtoAhpCorBSA(at A 414 ) were determined from the spectra. Intensities at the indicated time points were subtracted from the respective zero values and plotted. The presented curves are representative of three independent experiments.CC-BY · From the PDF

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  1. Nagababu, 2004, Antioxid. Redox. Signal., 6, 967
  2. Sassa, 2004, Antioxid. Redox. Signal., 6, 819

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Metadata

Authors
Lechardeur, D., Fernandez, A., Robert, B., Gaudu, P., Trieu-Cuot, P., Lamberet, G., & Gruss, A.
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2010-05
Volume
285
Issue
21
Pages
16032-16041
ISSN
0021-9258
DOI
10.1074/jbc.m109.024505
Cited by
47
References
72
Language
en
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