Addition of iron salts to chaotrope-denatured aporubredoxin (apoRd) leads to nearly quantitative recovery of its single Fe(SCys) 4 site and native protein structure without significant dilution of the chaotrope. This "high chaotrope" approach was used to examine iron binding and protein folding events using stopped-flow UV/vis absorption and CD spectroscopies. At 100-fold molar excess ferrous iron over denatured apoRd maintained in 5 M urea, the folded holoFe III Rd structure was recovered in >90% yield with t 1/2 < 10 msec. More modest excesses of iron also gave nearly quantitative holoRd formation in 5 M urea but with chronological resolution of iron binding and protein folding events. The results indicate structural recovery in 5 M urea consists of the minimal sequence: (1) binding of ferrous iron to the unfolded apoRd, (2) rapid formation of a near-native ferrous Fe(SCys) 4 site within a protein having no detectable secondary structure, (3) recovery of the ferrous Fe(SCys) 4 site chiral environment nearly concomitantly with (4) recovery of the native protein secondary structure. The rate of step 2 (and by inference, step 1) was not saturated even at 100-fold molar excess of iron. Analogous results obtained on Cys→Ser iron ligand variants support formation of an unfolded-Fe(SCys) 3 complex between steps 1 and 2, which we propose is the key nucleation event that pulls distal regions of the protein chain together. These results show that folding of chaotrope-denatured apoRd is iron-nucleated and driven by extraordinarily rapid formation of the Fe(SCys) 4 site from an essentially random coil apoprotein. This high chaotrope, multi-spectroscopic approach could clarify folding pathways of other [M(SCys) 3
Supporting Information AvailableNear UV-CD spectra of apo and holoRds in high urea, plots of midpoint urea denaturations of apoRds, spectral time courses of ferrous ammonium sulfate oxidations in 5 M urea, static far-UV CD spectra of apo and holoRds in high urea, semi-log plots of the time courses in Figure 3 and Figure 6, stopped-flow absorption spectral time course for apoRd +110-fold molar excess iron in 4.6 M urea. This material is available free of charge via the Internet at http://pubs.acs.org. 1 Abbreviations used: Rd, rubredoxin, apoRd, metal-free Rd; holoRd, iron-containing Rd in its native folded structure; Cp, Clostridium pasteurianum; CD, circular dichroism; wt, wild type; Tris-HCl, tris(hydroxymethyl)aminomethane-hydrochloride; CXXC, two cysteine residues (C) separated by two other residues, X, in the Rd amino acid sequence; C6S, C9S, C39S, C42S, Cys→Ser variant Rds; unfolded-Fe II (SCys) 4 , species exhibiting near-UV absorption characteristic of the native Fe II (SCys) 4 site but no CD signal for protein secondary structure; chiral-Fe II (SCys) 4 , species exhibiting near-UV absorption and CD signals characteristic of the native Fe II (SCys) 4 site; folded-Fe II (SCys) 4 , species exhibiting absorption and CD signals for the Fe II (SCys) 4 site and protein secondary structure CD signal char...