Slow formation of [3Fe-4S](1+) clusters in mutant forms of Desulfovibrio africanus ferredoxin III

Desulfovibrio africanus ferredoxia III (Da FdIII) readily interconverts bet ween a 7Fe and an 8Fe form with Asp-14 believed to provide a cluster ligand in the latter form. To investigate the factors important for cluster inter conversion in Fe/S cluster-containing proteins we have studied two variants of Da FdIII produced by site-directed mutagenesis, Asp14Glu and Asp14His, with cluster incorporation performed in vitro. Characterisation of these pr oteins by UV/visible, EPR and H-1 NMR spectroscopies revealed that the form ation of the stable 7Fe form of these proteins takes some time to occur. Ev idence is presented which indicates the [4Fe-4S](2+) cluster is incorporate d prior to the [3Fe-4S](1+) cluster. (C) 2000 Federation of European Bioche mical Societies.