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

Delphine Lechardeur,Annabelle Fernandez,Bruno Robert,Philippe Gaudu,Patrick Trieu-Cuot,Gilles Lamberet,Alexandra Gruss

doi:10.1074/jbc.m109.024505

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.

Highlights

Erichia coli and other heme-synthesizing bacteria, heme b is further processed to heme d by the activity of the heme hydroperoxidase II [9]; both heme forms are required for activity of the cytochrome bd oxidase (10 –12)
Among the proteins identified by MALDI-TOF mass spectrometry, a protein band migrating at around 20 kDa was identified as alkyl hydroxyperoxide reductase (AhpC)
In GBS and numerous bacteria, ahpC is clustered with ahpF, the gene coding for the alkyl hydroperoxide reductase (AhpF) subunit, which functions as an electron donor to regenerate AhpC [44]

Summary

Introduction

Erichia coli and other heme-synthesizing bacteria, heme b is further processed to heme d by the activity of the heme hydroperoxidase II (the catalase KatE) [9]; both heme forms are required for activity of the cytochrome bd oxidase (10 –12). ROS such as H2O2 or hydroperoxides can initiate nonenzymatic modification and degradation of heme [13, 14] In view of these data and the low solubility of free heme, it is probable that internalized heme does not exist as a free molecule in the cytoplasm but as a complex with hemebinding proteins, which would reduce its contact with the cellular environment and facilitate its intracellular trafficking. Our goal is to identify GBS proteins responsible for heme homeostasis. To this purpose, we used heme affinity. We present the characterization of one of these proteins, the ubiquitous alkyl hydroxyperoxide reductase (AhpC), and show that it is a heme-binding protein. In Helicobacter pylori, AhpC was shown to oligomerize and acquire protein chaperone activity in oxidative stress conditions [28]

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