title: Thermal stability of Clostridium pasteurianum rubredoxin: deconvoluting the contributions of the metal site and the protein.
abstract: To provide a framework for understanding the hyperthermostability of some rubredoxins, a comprehensive analysis of the thermally induced denaturation of rubredoxin (Rd) from the mesophile, Clostridium pasteurianum was undertaken. Rds with three different metals in its M(SCys)4 site (M = Fe3+/2+, Zn2+, or Cd2+) were examined. Kinetics of metal ion release were monitored anaerobically at several fixed temperatures between 40 and 100 degrees C, and during progressive heating of the iron-containing protein. Both methods gave a thermal stability of metal binding in the order Fe2+ << Fe3+ < Zn2+ < Cd2+. The temperature at which half of the iron was released from the protein in temperature ramp experiments was 69 degrees C for Fe2+ Rd and 83 degrees C for Fe3+ Rd. Temperature-dependent changes in the protein structure were monitored by differential scanning calorimetry, tryptophan fluorescence, binding of a fluorescent hydrophobic probe, and 1H NMR. Major but reversible structural changes, consisting of swelling of the hydrophobic core and opening of a loop region, were found to occur at temperatures (50-70 degrees C) much lower than those required for loss of the metal ion. For the three divalent metal ions, the results suggest that the onset of the reversible, lower-temperature structural changes is dependent on the size of the MS4 site, whereas the final, irreversible loss of metal ion is dependent on the inherent M-SCys bond strength. In the case of Fe3+ Rd, stoichiometric Fe3+/cysteine-ligand redox chemistry also occurs during metal ion loss. The results indicate that thermally induced unfolding of the native Cp Rd must surmount a significant kinetic barrier caused by stabilizing interactions both within the protein and within the M(SCys)4 site.
authors.affiliation: Dipartimento di Scienze Molecolari Agroalimentari, Università degli Studi di Milano, Milan, Italy. francesco.bonomi@unimi.it
authors.forename: F
authors.initials: F
authors.lastname: Bonomi
authors.forename: D
authors.initials: D
authors.lastname: Fessas
authors.forename: S
authors.initials: S
authors.lastname: Iametti
authors.forename: D M
authors.initials: DM
authors.lastname: Kurtz
authors.forename: S
authors.initials: S
authors.lastname: Mazzini
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002104:Cadmium / Q000378:metabolism; D003013:Clostridium / Q000737:chemistry*; D004355:Drug Stability; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D019216:Metals, Heavy / Q000378:metabolism; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012416:Rubredoxins / Q000737:chemistry* / Q000378:metabolism; D013057:Spectrum Analysis; D013696:Temperature; D015032:Zinc / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D019216:Metals, Heavy; D012416:Rubredoxins; D002104:Cadmium; D007501:Iron; D015032:Zinc