Wednesday, November 4, 2009: 9:20 AM
Kohlberg (Camino Real Hotel)
The iron-sulfur protein, rubredoxin (Rd), which contains a single Fe(SCys)4 site, has been the subject of numerous studies of thermal and denaturant-induced unfolding due to its small size (~54 residues) and extraordinary resistance to denaturation. However, our recent study1 is the only one describing the physiologically more relevant process of iron uptake and folding of apoRd leading to the native structure. We report here more extensive and detailed studies of this process, including an extraordinary observation: the apparently fully native protein structure, including the Fe(SCys)4 site, can be rapidly (≤10 msec) recovered in >90% yield upon addition of aqueous ferrous iron to the fully denatured apoRd at room temperature in 5 M urea without dilution of the denaturant! Rapid-mixing UV-vis absorbance and CD spectral time courses of wild-type apoRd and its Cys-to-Ser iron ligand variants were used to resolve the structural recovery in 5 M urea into the minimal sequence: (1) rapid binding of ferrous iron to the unfolded apoRd, (2) formation of a near-native ferrous Fe(SCys)4 site, and (3) recovery of the native secondary structure. The rate of step (2) (and by inference, step (1)) could not be saturated even at 100-fold molar excess of iron. The folding of apoRd in high urea is, thus, iron-nucleated, and, despite its extreme rapidity, cannot be described by a simple two-state, denatured-to-native folding process.
1. Bonomi, F., Iametti, S., Ferranti, P., Kurtz, D. M., Jr., Morleo, A., Ragg, E. M. J. Biol. Inorg. Chem. 2008, 13, 981-991.