A Two-component NADPH Oxidase (NOX)-like System in Bacteria Is Involved in the Electron Transfer Chain to the Methionine Sulfoxide Reductase MsrP

Céline Juillan-Binard,Antoine Picciocchi,Jean-Pierre Andrieu,Jerome Dupuy,Isabelle Petit-Hartlein,Christelle Caux-Thang,Corinne Vivès,Vincent Nivière,Franck Fieschi

doi:10.1074/jbc.m116.752014

Abstract

MsrPQ is a newly identified methionine sulfoxide reductase system found in bacteria, which appears to be specifically involved in the repair of periplasmic proteins oxidized by hypochlorous acid. It involves two proteins: a periplasmic one, MsrP, previously named YedY, carrying out the Msr activity, and MsrQ, an integral b-type heme membrane-spanning protein, which acts as the specific electron donor to MsrP. MsrQ, previously named YedZ, was mainly characterized by bioinformatics as a member of the FRD superfamily of heme-containing membrane proteins, which include the NADPH oxidase proteins (NOX/DUOX). Here we report a detailed biochemical characterization of the MsrQ protein from Escherichia coli We optimized conditions for the overexpression and membrane solubilization of an MsrQ-GFP fusion and set up a purification scheme allowing the production of pure MsrQ. Combining UV-visible spectroscopy, heme quantification, and site-directed mutagenesis of histidine residues, we demonstrated that MsrQ is able to bind two b-type hemes through the histidine residues conserved between the MsrQ and NOX protein families. In addition, we identify the E. coli flavin reductase Fre, which is related to the dehydrogenase domain of eukaryotic NOX enzymes, as an efficient cytosolic electron donor to the MsrQ heme moieties. Cross-linking experiments as well as surface Plasmon resonance showed that Fre interacts with MsrQ to form a specific complex. Taken together, these data support the identification of the first prokaryotic two-component protein system related to the eukaryotic NOX family and involved in the reduction of periplasmic oxidized proteins.

Highlights

MsrQ Overexpression, Solubilization, and Purification—The optimization of MsrQ production in E. coli was performed following a standardized protocol, which has allowed the overproduction of many E. coli MG1655 membrane proteins fused to GFP [18]
For each of these conditions, the GFP fluorescence was measured on the whole bacterial cell population to assess the MsrQ-GFP production level
MsrQ belongs to an enzymatic system involving MsrP (YedY), a new type of methionine sulfoxide reductase, which repairs periplasmic methionine sulfoxide-containing proteins

Summary

Introduction

A new type of Msr has been discovered in Gram-negative bacteria [2, 3] This system appears to be involved in the repair of periplasmic proteins oxidized by hypochlorous acid HOCl. HOCl is generated in particular within phagocytic cells of the mammalian innate immune system to kill internalized pathogens. Long before the identification of its physiological function, bioinformatic studies recognized MsrQ as an archetypal domain present in an important superfamily of heme-containing membrane proteins initially designated as the YedZ family [11] This domain, present in bacterial and eukaryotic representatives, has been renamed as the FRD domain (for ferric reductase domain) [12], based on its identification within STEAP (six-transmembrane epithelial antigen of the prostate). The FRD domain is present within the NADPH oxidase (NOX/ DUOX) family of transmembrane enzymes involved in the production of reactive oxygen species (ROS)3 [16]

Methods

Results

Conclusion