title: Structural Bases for the Regulation of CO Binding in the Archaeal Protoglobin from Methanosarcina acetivorans.
abstract: Studies of CO ligand binding revealed that two protein states with different ligand affinities exist in the protoglobin from Methanosarcina acetivorans (in MaPgb*, residue Cys(E20)101 was mutated to Ser). The switch between the two states occurs upon the ligation of MaPgb*. In this work, site-directed mutagenesis was used to explore the role of selected amino acids in ligand sensing and stabilization and in affecting the equilibrium between the "more reactive" and "less reactive" conformational states of MaPgb*. A combination of experimental data obtained from electronic and resonance Raman absorption spectra, CO ligand-binding kinetics, and X-ray crystallography was employed. Three amino acids were assigned a critical role: Trp(60)B9, Tyr(61)B10, and Phe(93)E11. Trp(60)B9 and Tyr(61)B10 are involved in ligand stabilization in the distal heme pocket; the strength of their interaction was reflected by the spectra of the CO-ligated MaPgb* and by the CO dissociation rate constants. In contrast, Phe(93)E11 is a key player in sensing the heme-bound ligand and promotes the rotation of the Trp(60)B9 side chain, thus favoring ligand stabilization. Although the structural bases of the fast CO binding rate constant of MaPgb* are still unclear, Trp(60)B9, Tyr(61)B10, and Phe(93)E11 play a role in regulating heme/ligand affinity.
authors.affiliation: Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium.
authors.forename: Lesley
authors.initials: L
authors.lastname: Tilleman
authors.affiliation: Department of Physics and Earth Sciences, University of Parma, Parma, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Abbruzzetti
authors.affiliation: Department of Clinical Sciences and Translational Medicine, University of Roma Tor Vergata, Roma, Italy; Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, Bari, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Ciaccio
authors.affiliation: Department of Clinical Sciences and Translational Medicine, University of Roma Tor Vergata, Roma, Italy.
authors.forename: Giampiero
authors.initials: G
authors.lastname: De Sanctis
authors.affiliation: Department of Biosciences, University of Milano, Milano, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Nardini
authors.affiliation: Department of Physics, University of Genova, Genova, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Pesce
authors.affiliation: Department of Physics, University of Antwerp, Antwerp, Belgium.
authors.forename: Filip
authors.initials: F
authors.lastname: Desmet
authors.affiliation: Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium.
authors.forename: Luc
authors.initials: L
authors.lastname: Moens
authors.affiliation: Department of Physics, University of Antwerp, Antwerp, Belgium.
authors.forename: Sabine
authors.initials: S
authors.lastname: Van Doorslaer
authors.affiliation: Department of Pharmacy, University of Parma, Parma, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Bruno
authors.affiliation: Department of Biosciences, University of Milano, Milano, Italy.
authors.forename: Martino
authors.initials: M
authors.lastname: Bolognesi
authors.affiliation: Interdepartmental Laboratory of Electron Microscopy, University Roma Tre, Roma, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Ascenzi
authors.affiliation: Department of Clinical Sciences and Translational Medicine, University of Roma Tor Vergata, Roma, Italy; Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, Bari, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Coletta
authors.affiliation: Department of Physics and Earth Sciences, University of Parma, Parma, Italy.
authors.forename: Cristiano
authors.initials: C
authors.lastname: Viappiani
authors.affiliation: Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium.
authors.forename: Sylvia
authors.initials: S
authors.lastname: Dewilde
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002248:Carbon Monoxide / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D007700:Kinetics; D008024:Ligands; D017020:Methanosarcina / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010782:Photolysis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman
chemical_list: D019843:Archaeal Proteins; D008024:Ligands; D011994:Recombinant Proteins; D002248:Carbon Monoxide