title: Dynamic features of the subunit interface of Cu,Zn superoxide dismutase as probed by tryptophan phosphorescence.
abstract: As part of the more general inquiry on the molecular basis of specific recognition between macromolecules, the subunit-subunit interface structure of dimeric superoxide dismutase from Photobacterium leiognathi has been probed selectively by the phosphorescence emission of Trp-73, located at the subunit contact region. Copper at the catalytic site was found to quench completely the delayed emission and therefore all studies were conducted with the copper-free or Cd(2+)-substituted protein. The spectrum at 140 K is diagnostic for an indole ring located in a hydrophobic environment whereas a degree of spectral broadening indicates that the local structure is not unique. Environmental heterogeneity is confirmed by the nonuniform phosphorescence decay in buffer, at 274 K, with lifetime components of 44 and 20 ms of practically equal amplitude. Information on the flexibility of the interface region was gathered from both the intrinsic lifetime and the accessibility of acrylamide to the site of the chromophore. The magnitude of the intrinsic lifetime, its temperature dependence, and the accessibility to solutes like acrylamide describe a tight dimeric structure in which hydrophobic interactions seem to play an important role. In particular the acrylamide bimolecular rate constant is 1.4 x 10(4) M(-1) s(-1) and indicates highly hindered diffusion of the solute through the interface region. Cd(2+) complexation to the apoprotein caused no detectable changes in protein conformation although the metal was able to influence the flexibility of the Trp-73 environment, indicating the occurrence of a long-range communication between the intersubunit surface and the active site, which is more than 16 A away.
authors: Istituto di Biofisica Consiglio Nazionale delle Ricerche Area della Ricerca di Pisa, Via G. Moruzzi, 1, Pisa, Italy. patrizia.cioni@ib.pi.cnr.it
authors: P
authors: P
authors: Cioni
authors: M E
authors: ME
authors: Stroppolo
authors: A
authors: A
authors: Desideri
authors: G B
authors: GB
authors: Strambini
mesh_terms: D003300:Copper / Q000737:chemistry* / Q000172:deficiency; D004926:Escherichia coli; D005456:Fluorescent Dyes / Q000737:chemistry; D008163:Luminescent Measurements; D010776:Photobacterium / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D003300:Copper; D014364:Tryptophan; D013482:Superoxide Dismutase