Formation of linear three-iron clusters in Aquifex aeolicus two-iron ferredoxins: effect of protein-unfolding speed

Abstract

The presence of a linear [3Fe–4S] cluster in a protein was first observed in beef-heart aconitase . Here, we report the formation of linear [3Fe–4S] clusters upon guanidine hydrochloride (GuHCl)-induced unfolding of Aquifex aeolicus [2Fe–2S] ferredoxins (Fd) ( Aae Fd1, Aae Fd4, and Aae Fd5) at alkaline conditions (pH 10, 20 °C). We find the mechanism of linear [3Fe–4S] cluster formation to depend critically on the speed of polypeptide unfolding. In similarity to seven-iron Fds, polypeptide unfolding determines the rate by which linear [3Fe–4S] clusters form in Aae Fd4 and Aae Fd5. In contrast, in a disulfide-lacking variant of Aae Fd1, which unfolds faster than Aae Fd4 and Aae Fd5, the polypeptides unfold first and the majority of clusters decompose. Next, unfolded polypeptides retaining intact clusters scavenge iron and sulfur to form linear [3Fe–4S] clusters in a bimolecular reaction. Wild-type Aae Fd1 unfolds slower than the speed of linear-cluster decomposition, and the linear species is never populated. Linear [3Fe–4S] clusters may be intermediates during folding of iron–sulfur proteins.