A step toward understanding the folding mechanism of bovine adrenodoxin

The iron-sulfur clusters of iron-sulfur proteins are not only essential for the structure and function but they also seem to play an important role in the folding process of these proteins. So far, no data on reversible unfol ding/refolding of iron-sulfur proteins under aerobic conditions have been r eported. We found appropriate conditions, which might also be applicable fo r other iron-sulfur proteins, for reversible unfolding/refolding of bovine adrenodoxin (Adx) that prevent cluster decomposition during the unfolding p rocess. The unfolding/refolding studies have been performed under aerobic c onditions using fluorescence measurements (with mutant Y82W of Adx, providi ng a sensitive internal probe), absorption, and circular dichroism (CD) spe ctroscopy as well as activity measurements. Without protecting reagent, adr enodoxin becomes an apoprotein upon denaturation which is an irreversible p rocess with respect to cluster rebinding. However, reversibility of unfoldi ng/refolding can be observed after protein denaturation in the presence of dithiothreitol (DTT), Upon removal of the denaturant, we regained 65, 63, a nd 64% refolding from CD, fluorescence, and activity measurements, respecti vely, In the case of thermal denaturation, the percentage of refolding is a bout 60% according to CD measurements. DTT appears to stabilize the [2Fe-2S ] cluster and prevents its decomposition during aerobic unfolding, providin g thereby the means of correct refolding of the protein. (C) 1999 Academic Press.