Iron-nucleated folding of a metalloprotein in high urea: resolution of metal binding and protein folding events

Abstract

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. With a 100-fold molar excess of ferrous iron over denatured apoRd maintained in 5 M urea, the folded holoFe(III)Rd structure was recovered in >90% yield with a t(1/2) of <10 ms. 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, and (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 a 100-fold molar excess of iron. Analogous results obtained for 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 together distal regions of the protein chain. 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, multispectroscopy approach could clarify folding pathways of other [M(SCys)(3)]- or [M(SCys)(4)]-containing proteins.

Figure 1

Cp holoRd structural representations. (Left) backbone with iron site shown as a sphere, Cys ligands as sticks with sequence numbers indicated, and backbone N-H---SCys hydrogen bonds indicated by dashed lines. Backbone is color spectrum-traced from blue at the N-terminus to red at the C-terminus. (Right) space-filling view (hydrogen atoms omitted). Drawings were generated using PyMOL (http://www.pymol.org) and coordinates from PDB ID 1IRO.

Figure 2

UV-vis absorption spectral time courses upon anaerobic stopped-flow mixing 10 volumes of 55 µM apoRd in buffer + 5 M urea with 1 volume of either 0.6 (1.1× Fe) or 6 mM (11× Fe) ferrous ammonium sulfate in buffer. For the 10-sec time courses, spectra were obtained from 100 msec to 10 sec at 100-msec intervals following the mixing dead time (~3 msec). Arrows indicate directions of absorbance changes.

Figure 3

Single-wavelength time courses for the spectral signal recoveries upon anaerobic stopped-flow mixing of apoRd in 5 M urea with ferrous ammonium sulfate as described in the legend to Figure 2. Background iron oxidation time courses have been subtracted where appropriate.

Figure 4

Static UV-vis absorption (top panel) and visible CD (bottom panel) spectra following anaerobic stopped-flow mixing 10 volumes of 55 µM wt, C9S or C42S apoRd in buffer + 5 M urea with 1 volume of 0.6 mM of ferrous ammonium sulfate in buffer (1.1-fold molar excess of iron over protein). Wt apoRd spectra without added iron are shown for comparison.

Figure 5

UV-vis absorption spectral time courses upon anaerobic stopped-flow mixing of C9S or C42S apoRd in 5 M urea with ferrous ammonium sulfate as described in the Figure 4 legend. Spectra were obtained from 3 s to 300 s at 3-sec intervals following the mixing dead time (~3 msec). Arrows indicate direction of absorbance changes.

Figure 6

Single-wavelength time courses for spectral signal recoveries upon anaerobic stopped-flow mixing of C9S or C42S apoRds in 5 M urea with ferrous ammonium sulfate as described in the Figure 4 legend. Wavelengths used to monitor signal recoveries are listed in the Table 2 footnote. Background iron oxidation time courses have been subtracted where appropriate.

Figure 7

A schematic model for the sequential Cys ligation of denatured apoRd leading to the unfolded-Fe^II(SCys)₄ in high urea. The protein chains are not depicted to uniform scale. Iron is represented as a sphere. The denatured-apo likely consists of an ensemble of fluxional structures. The choice of the Cys6/9 rather than the Cys39/42 residue pair as the initial iron chelate is arbitrary.