Abstract
The relationship between iron uptake by aporubredoxins (apoRds) and formation of native holorubredoxins (holoRd), including their Fe(SCys)4 sites, was studied. In the absence of denaturants, apoRds exhibited spectroscopic features consistent with structures very similar to those of the folded holoRds. However, additions of either ferric or ferrous salts to the apoRds in the absence of denaturants gave less than 40% recovery of the native holoRd circular dichroism and UV–vis spectroscopic features. In the presence of either 6 M urea or 6 M guanidine hydrochloride, the nativelike structural features of the apoRds were absent. Nevertheless, nearly quantitative recoveries of the native holoRd spectroscopic features were achieved by addition of either ferric or ferrous salts to the denatured apoRds without diluting the denaturant. Consistent with this observation, the native spectroscopic features were unaffected by addition of the same denaturant concentrations to the as-isolated holoRds. Denaturing concentrations of urea or guanidine hydrochloride also increased the rates of holoRd recoveries from apoRds and ferrous salts. Mass spectrometry confirmed that ferric iron binding to the denatured apoRds precedes the recoveries of protein secondary structures and Fe(SCys)4 sites. Thus, iron binding to the apoRds guides, both kinetically and thermodynamically, refolding to the native holoRd structures. Our results imply that the ferrous oxidation state would more efficiently drive formation of the native holoRd structure from the nascent apoprotein in vivo, but that the Fe(SCys)4 site must attain the ferric state in order to achieve its native structure.
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Abbreviations
- CD:
-
Circular dichroism
- Cp:
-
Clostridium pasteurianum
- ESI:
-
Electrospray ionization
- GuHCl:
-
Guanidine hydrochoride
- MS:
-
Mass spectrometry
- Pf:
-
Pyrococcus furiosus
- Rd:
-
Rubredoxin
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Acknowledgments
This work was supported in part by funds from the University of Milan (FIRST, to F.B.) and US National Institutes of Health grant GM040388 (to D.M.K.). A.M. is the grateful recipient of a postdoctoral fellowship from the University of Milan.
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Bonomi, F., Iametti, S., Ferranti, P. et al. “Iron priming” guides folding of denatured aporubredoxins. J Biol Inorg Chem 13, 981–991 (2008). https://doi.org/10.1007/s00775-008-0385-4
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DOI: https://doi.org/10.1007/s00775-008-0385-4