Site-directed mutants of adrenodoxin were studied for their ability to undergo cluster-iron substitution when reacted with zinc or cadmium salts under non-denaturing conditions in the presence or absence of reductants. Equilibrium and kinetic data for metal substitution were correlated with data on the stability to thermal unfolding and with the redox potential of the protein. Similarly to the wild-type protein, all mutants were able to stabilize a substituted form of the protein containing two metal (Zn or Cd) atoms and two sulfide ions/mol protein and a substituted form of the protein containing two sulfide ions and five Cd atoms/mol protein. However, the distribution of these two metal-substituted forms was different among the investigated proteins. [Ser95]Adrenodoxin stabilized either metal-substituted forms, confirming that Cys95 is not involved in metal coordination, even when five Cd atoms are bound to the protein. Removal of the extremely conserved hydroxy function at position 54 resulted in complete apoprotein formation upon reaction with Cd (75 % with Zn) under reducing conditions, indicating a cluster-harboring role for this function, which is conserved in all known 2Fe-2S proteins. Mutants at His56, which represents a residue unique to most vertebrate-type ferredoxins, were much more reactive than the wild-type protein with either metal, indicating that His56 plays a prominent role in the stabilization of the protein structure in the immediate vicinity of the cluster in this class of proteins. The nature of the metal-substitution products was dependent on cluster accessibility. For the reduced proteins, apoprotein formation depended on protein stability, while the velocity of metal substitution depended on the ease of cluster reduction.
Cluster-iron substitution is related to structural and functional features of adrenodoxin mutants and to their redox states / S. Iametti, H. Uhlmann, E. Ragg, N. Sala, A. Grinberg, V. Beckert, R. Bernhardt, F. Bonomi. - In: EUROPEAN JOURNAL OF BIOCHEMISTRY. - ISSN 0014-2956. - 251:3(1998 Feb 01), pp. 673-681.
|Titolo:||Cluster-iron substitution is related to structural and functional features of adrenodoxin mutants and to their redox states|
IAMETTI, STEFANIA (Primo)
BONOMI, FRANCESCO (Ultimo)
|Parole Chiave:||Adrenodoxin; Adrenodoxin mutant; Metal substitution; Redox potential; Thermal stability|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
Settore CHIM/06 - Chimica Organica
|Data di pubblicazione:||1-feb-1998|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1046/j.1432-1327.1998.2510673.x|
|Appare nelle tipologie:||01 - Articolo su periodico|