Redox Behavior of the S-Adenosylmethionine (SAM)-Binding Fe-S Cluster in Methylthiotransferase RimO, toward Understanding Dual SAM Activity.
RimO, a radical-S-adenosylmethionine (SAM) enzyme, catalyzes the specific C3methylthiolation of the D89 residue in the ribosomal S12protein. Two intact iron-sulfur clusters and two SAM cofactors both are required for catalysis. By using electron paramagnetic resonance, Mössbauer spectroscopies, and site-directed mutagenesis, we show how two SAM molecules sequentially bind to the unique iron site of the radical-SAM cluster for two distinct chemical reactions in RimO. Our data establish that the two SAM molecules bind the radical-SAM cluster to the unique iron site, and spectroscopic evidence obtained under strongly reducing conditions supports a mechanism in which the first molecule of SAM causes the reoxidation of the reduced radical-SAM cluster, impeding reductive cleavage of SAM to occur and allowing SAM to methylate a HS-ligand bound to the additional cluster. Furthermore, by using density functional theory-based methods, we provide a description of the reaction mechanism that predicts the attack of the carbon radical substrate on the methylthio group attached to the additional [4Fe-4S] cluster.
Laboratoire de Chimie et Biologie des Métaux, team "Biocatalyse", Biosciences & Biotechnology Institute of Grenoble (BIG), BIG/LCBM/Biocat, UMR 5249 CEA/CNRS/UGA, CEA/Grenoble, 17, rue des Martyrs, Grenoble, France.
Thibaut
T
Molle
Laboratoire de Chimie et Biologie des Métaux, team "MCT" Biosciences & Biotechnology Institute of Grenoble (BIG), BIG/LCBM/MCT, UMR 5249 CEA/CNRS/UGA, CEA/Grenoble, 17, rue des Martyrs, Grenoble, France.
Yohann
Y
Moreau
Laboratoire de Chimie et Biologie des Métaux, team "PMB" Biosciences & Biotechnology Institute of Grenoble (BIG), BIG/LCBM/PMB, UMR 5249 CEA/CNRS/UGA, CEA/Grenoble, 17, rue des Martyrs, Grenoble, France.
Martin
M
Clemancey
Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University , New York, New York 10027, United States.
Farhad
F
Forouhar
University Grenoble Alpes , INAC-SCIB, F-38000 Grenoble, France.|CEA , INAC-SyMMES, F-38000 Grenoble, France.
Jean-Luc
JL
Ravanat
Laboratoire de Chimie et Biologie des Métaux, team "Affond" Biosciences & Biotechnology Institute of Grenoble (BIG), BIG/LCBM/Affond, UMR 5249 CEA/CNRS/UGA, CEA/Grenoble, 17, rue des Martyrs, Grenoble, France.
Nicolas
N
Duraffourg
Aix-Marseille University , CNRS, BIP UMR 7281, 31 chemin J. Aiguier, 13402 Marseille Cedex 20, France.
Vincent
V
Fourmond
Laboratoire de Chimie et Biologie des Métaux, team "PMB" Biosciences & Biotechnology Institute of Grenoble (BIG), BIG/LCBM/PMB, UMR 5249 CEA/CNRS/UGA, CEA/Grenoble, 17, rue des Martyrs, Grenoble, France.
Jean-Marc
JM
Latour
University Grenoble Alpes, INAC, SCIB/LRM , F-38000 Grenoble, France.|CEA, INAC, SCIB/LRM, F-38054 Grenoble, France.
Serge
S
Gambarelli
Laboratoire de Chimie et Biologie des Métaux, team "Biocatalyse", Biosciences & Biotechnology Institute of Grenoble (BIG), BIG/LCBM/Biocat, UMR 5249 CEA/CNRS/UGA, CEA/Grenoble, 17, rue des Martyrs, Grenoble, France.
Etienne
E
Mulliez
Laboratoire de Chimie et Biologie des Métaux, team "Biocatalyse", Biosciences & Biotechnology Institute of Grenoble (BIG), BIG/LCBM/Biocat, UMR 5249 CEA/CNRS/UGA, CEA/Grenoble, 17, rue des Martyrs, Grenoble, France.
Mohamed
M
Atta
D002384:Catalysis; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D012436:S-Adenosylmethionine / Q000378:metabolism*; D013057:Spectrum Analysis / Q000379:methods; D013466:Sulfurtransferases / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; D012436:S-Adenosylmethionine; C545011:RimO protein, E coli; D013466:Sulfurtransferases