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Addressing Ligand-Based Redox in Molybdenum-Dependent Methionine Sulfoxide Reductase.

ABSTRACT: A combination of pulsed EPR, CW EPR, and X-ray absorption spectroscopies has been employed to probe the geometric and electronic structure of the E. coli periplasmic molybdenum-dependent methionine sulfoxide reductase (MsrP). ¹⁷O and ¹H pulsed EPR spectra show that the as-isolated Mo(V) enzyme form does not possess an exchangeable H₂O/OH^- ligand bound to Mo as found in the sulfite oxidizing enzymes of the same family. The nature of the unusual CW EPR spectrum has been re-evaluated in light of new data on the MsrP-N45R variant and related small-molecule analogues of the active site. These data point to a novel "thiol-blocked" [(PDT)Mo^VO(S_Cys)(thiolate)]^- structure, which is supported by new EXAFS data. We discuss these new results in the context of ligand-based and metal-based redox chemistry in the enzymatic oxygen atom transfer reaction.

SUBMITTER: Ingersol LJ

PROVIDER: S-EPMC7937415 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Addressing Ligand-Based Redox in Molybdenum-Dependent Methionine Sulfoxide Reductase.

Ingersol Laura J LJ Yang Jing J Kc Khadanand K Pokhrel Amrit A Astashkin Andrei V AV Weiner Joel H JH Johnston Christopher A CA Kirk Martin L ML

Journal of the American Chemical Society 20200128 6

A combination of pulsed EPR, CW EPR, and X-ray absorption spectroscopies has been employed to probe the geometric and electronic structure of the E. coli periplasmic molybdenum-dependent methionine sulfoxide reductase (MsrP). 17O and 1H pulsed EPR spectra show that the as-isolated Mo(V) enzyme form does not possess an exchangeable H2O/OH- ligand bound to Mo as found in the sulfite oxidizing enzymes of the same family. The nature of the un ...[more]

PMID: 31989824

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Corynebacterium diphtheriae methionine sulfoxide reductase a exploits a unique mycothiol redox relay mechanism.

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Methionine sulfoxide reductase A is a stereospecific methionine oxidase.

Project description:Methionine sulfoxide reductase A (MsrA) catalyzes the reduction of methionine sulfoxide to methionine and is specific for the S epimer of methionine sulfoxide. The enzyme participates in defense against oxidative stresses by reducing methionine sulfoxide residues in proteins back to methionine. Because oxidation of methionine residues is reversible, this covalent modification could also function as a mechanism for cellular regulation, provided there exists a stereospecific methionine oxidase. We show that MsrA itself is a stereospecific methionine oxidase, producing S-methionine sulfoxide as its product. MsrA catalyzes its own autooxidation as well as oxidation of free methionine and methionine residues in peptides and proteins. When functioning as a reductase, MsrA fully reverses the oxidations which it catalyzes.

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Myristoylated methionine sulfoxide reductase A is a late endosomal protein.

Project description:Methionine residues in proteins provide antioxidant defense by reacting with oxidizing species, which oxidize methionine to methionine sulfoxide. Reduction of the sulfoxide back to methionine is catalyzed by methionine sulfoxide reductases, essential for protection against oxidative stress. The nonmyristoylated form of methionine sulfoxide reductase A (MSRA) is present in mitochondria, whereas the myristoylated form has been previously reported to be cytosolic. Despite the importance of MSRA in antioxidant defense, its in vivo binding partners and substrates have not been identified. Starting with a protein array, and followed by immunoprecipitation experiments, colocalization studies, and subcellular fractionation, we identified the late endosomal protein, StAR-related lipid transfer domain-containing 3 (STARD3), as a binding partner of myristoylated MSRA, but not of nonmyristoylated MSRA. STARD3 is known to have both membrane-binding and cytosolic domains that are important in STARD3-mediated transport of cholesterol from the endoplasmic reticulum to the endosome. We found that the STARD3 cytosolic domain localizes MSRA to the late endosome. We propose that the previous conclusion that myristoylated MSRA is strictly a cytosolic protein is artifactual and likely due to vigorous overexpression of MSRA. We conclude that myristoylated MSRA is a late endosomal protein that may play a role in lipid metabolism or may protect endosomal proteins from oxidative damage.

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Structural and biochemical analysis of mammalian methionine sulfoxide reductase B2.

Project description:Methionine sulfoxide reductases are antioxidant enzymes that repair oxidatively damaged methionine residues in proteins. Mammals have three members of the methionine-R-sulfoxide reductase family, including cytosolic MsrB1, mitochondrial MsrB2, and endoplasmic reticulum MsrB3. Here, we report the solution structure of reduced Mus musculus MsrB2 using high resolution nuclear magnetic resonance (NMR) spectroscopy. MsrB2 is a ?-strand rich globular protein consisting of eight antiparallel ?-strands and three N-terminal ?-helical segments. The latter secondary structure elements represent the main structural difference between mammalian MsrB2 and MsrB1. Structural comparison of mammalian and bacterial MsrB structures indicates that the general topology of this MsrB family is maintained and that MsrB2 more resembles bacterial MsrBs than MsrB1. Structural and biochemical analysis supports the catalytic mechanism of MsrB2 that, in contrast to MsrB1, does not involve a resolving cysteine (Cys). pH dependence of catalytically relevant residues in MsrB2 was accessed by NMR spectroscopy and the pK(a) of the catalytic Cys162 was determined to be 8.3. In addition, the pH-dependence of MsrB2 activity showed a maximum at pH 9.0, suggesting that deprotonation of the catalytic Cys is a critical step for the reaction. Further mobility analysis showed a well-structured N-terminal region, which contrasted with the high flexibility of this region in MsrB1. Our study highlights important structural and functional aspects of mammalian MsrB2 and provides a unifying picture for structure-function relationships within the MsrB protein family.

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Structure of Mycobacterium tuberculosis methionine sulfoxide reductase A in complex with protein-bound methionine.

Project description:Peptide methionine sulfoxide reductase (MsrA) repairs oxidative damage to methionine residues arising from reactive oxygen species and reactive nitrogen intermediates. MsrA activity is found in a wide variety of organisms, and it is implicated as one of the primary defenses against oxidative stress. Disruption of the gene encoding MsrA in several pathogenic bacteria responsible for infections in humans results in the loss of their ability to colonize host cells. Here, we present the X-ray crystal structure of MsrA from the pathogenic bacterium Mycobacterium tuberculosis refined to 1.5 A resolution. In contrast to the three catalytic cysteine residues found in previously characterized MsrA structures, M. tuberculosis MsrA represents a class containing only two functional cysteine residues. The structure reveals a methionine residue of one MsrA molecule bound at the active site of a neighboring molecule in the crystal lattice and thus serves as an excellent model for protein-bound methionine sulfoxide recognition and repair.

| S-EPMC164888 | biostudies-literature

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