Unfolding and stability properties of iron-sulfur ferredoxins from the hyperthermophilic bacterium Aquifex aeolicus
Description
Cofactors are essential for biological function of many proteins. Since cofactors often can interact with both folded and unfolded proteins, they may also modulate protein structural changes. Iron-sulfur clusters are common cofactors that facilitate electron transport in ferredoxins. Proteins in organisms living at extreme temperatures often have mesophilic homologues that are very similar in structure, and the origin of thermostability is not obvious. Three ferredoxins with [2Fe-2S] clusters from hyperthermophilic Aquifex aeolicus (Aae, growth temperature 85°C) have been isolated and are studied in this work. AaeFd1, with a unique disulfide bond, and AaeFd5 both have the common ferredoxin-like fold whereas AaeFd4 has a thioredoxin-like fold To investigate the apparent stability and unfolding mechanisms of these ferredoxins, the native states were perturbed by heat and chemical denaturants, and the reactions were monitored by far-UV circular dichroism (secondary structure), fluorescence (fluorophore environment), visible absorption (cluster integrity), and calorimetry (heat changes). Unfolding of all Aquifex aeolicus ferredoxins at 20°C (pH 7) occurs in an apparent two-state process that requires high chemical denaturant concentrations and long incubation times. Thermally-induced unfolding of these ferredoxins requires temperatures above 100°C (pH 7). Comparisons of AaeFd1 and AaeFd1 C87A (variant in which one cysteine involved in the disulfide bond is altered to alanine) unfolding processes indicate that the disulfide bond contributes to the high stability of AaeFd1.Thermally- and chemically-induced unfolding of the dimeric AaeFd4 is protein concentration dependent, implying that dissociation is coupled to unfolding New visible absorption bands at 520 and 610nm appear transiently during high-pH unfolding of AaeFd1 C87A, AaeFd5, and AaeFd4. These spectroscopic features are characteristic of linear [3Fe-4S] clusters. This is the first time that [2Fe-2S] clusters have been found to rearrange into linear [3Fe-4S] clusters in structurally perturbed ferredoxins. The formation mechanisms for linear [3Fe-4S] clusters in the Aquifex aeolicus ferredoxins have been characterized: polypeptide unfolding speed plays a crucial role. Wild-type AaeFd1 does not form the linear [3Fe-4S] cluster since unfolding is slower than linear [3Fe-4S] cluster degradation. The in vitro observations of iron-sulfur clusters interacting with unfolded ferredoxins suggest that cofactor-binding may occur before ferredoxin folding in vivo