Formation of linear three-iron clusters in Aquifex aeolicus two-iron ferredoxins: effect of protein-unfolding speed.
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) (AaeFd1, AaeFd4, and AaeFd5) at alkaline conditions (pH 10, 20 degrees 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 AaeFd4 and AaeFd5. In contrast, in a disulfide-lacking variant of AaeFd1, which unfolds faster than AaeFd4 and AaeFd5, 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 AaeFd1 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.
Higgins
Catherine L
CL
Department of Chemistry, Tulane University, New Orleans, LA 70118, USA.
Wittung-Stafshede
Pernilla
P
D001426:Bacterial Proteins / Q000737:chemistry*; D005288:Ferredoxins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry*; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D007700:Kinetics; D009038:Motion; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding
D001426:Bacterial Proteins; D005288:Ferredoxins; D007506:Iron-Sulfur Proteins; D007501:Iron