Unanticipated coordination of tris buffer to the Radical SAM cluster of the RimO methylthiotransferase.
Radical SAM enzymes generally contain a [4Fe-4S](2+/1+) (RS cluster) cluster bound to the protein via the three cysteines of a canonical motif CxxxCxxC. The non-cysteinyl iron is used to coordinate SAM via its amino-carboxylate moiety. The coordination-induced proximity between the cluster acting as an electron donor and the adenosyl-sulfonium bond of SAM allows for the homolytic cleavage of the latter leading to the formation of the reactive 5'-deoxyadenosyl radical used for substrate activation. Most of the structures of Radical SAM enzymes have been obtained in the presence of SAM, and therefore, little is known about the situation when SAM is not present. In this report, we show that RimO, a methylthiotransferase belonging to the radical SAM superfamily, binds a Tris molecule in the absence of SAM leading to specific spectroscopic signatures both in Mössbauer and pulsed EPR spectroscopies. These data provide a cautionary note for researchers who work with coordinative unsaturated iron sulfur clusters.
Laboratoire de Chimie et Biologie des Métaux, team "Biocatalyse", Biosciences and 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 "Physicochimie des Métaux en Biologie" Biosciences and Biotechnology Institute of Grenoble (BIG), BIG-LCBM/PMB, UMR 5249 CEA/CNRS/UGA, CEA/Grenoble, 17, rue des Martyrs, Grenoble, France.
Martin
M
Clémancey
Laboratoire de Chimie et Biologie des Métaux, team "Physicochimie des Métaux en Biologie" Biosciences and 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, 38000, Grenoble, France.|CEA, INAC, SCIB/LRM, 38054, Grenoble, France.
Velavan
V
Kathirvelu
University Grenoble Alpes, INAC, SCIB/LRM, 38000, Grenoble, France.|CEA, INAC, SCIB/LRM, 38054, Grenoble, France.
Giuseppe
G
Sicoli
Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, NY, 10027, USA.
Farhad
F
Forouhar
Laboratoire de Chimie et Biologie des Métaux, team "Biocatalyse", Biosciences and Biotechnology Institute of Grenoble (BIG), BIG-LCBM/Biocat, UMR 5249 CEA/CNRS/UGA, CEA/Grenoble, 17, rue des Martyrs, Grenoble, France. etienne.mulliez@cea.fr.
Etienne
E
Mulliez
University Grenoble Alpes, INAC, SCIB/LRM, 38000, Grenoble, France. serge.gambarelli@cea.fr.|CEA, INAC, SCIB/LRM, 38054, Grenoble, France. serge.gambarelli@cea.fr.
Serge
S
Gambarelli
Laboratoire de Chimie et Biologie des Métaux, team "Biocatalyse", Biosciences and Biotechnology Institute of Grenoble (BIG), BIG-LCBM/Biocat, UMR 5249 CEA/CNRS/UGA, CEA/Grenoble, 17, rue des Martyrs, Grenoble, France. mohamed.atta@cea.fr.
Mohamed
M
Atta
D002021:Buffers; D012436:S-Adenosylmethionine / Q000737:chemistry* / Q000378:metabolism; D013466:Sulfurtransferases / Q000737:chemistry* / Q000378:metabolism; D020124:Thermotoga maritima / Q000201:enzymology; D014325:Tromethamine / Q000737:chemistry*
D002021:Buffers; D014325:Tromethamine; D012436:S-Adenosylmethionine; D013466:Sulfurtransferases
Biosynthesis; Cofactor; Electron paramagnetic resonance; Iron–sulfur cluster; Ligand binding; Mössbauer spectroscopy; Pulsed EPR