“Iron priming” guides folding of denatured aporubredoxins

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.Correspondence to: Francesco Bonomi. Electronic supplementary material The online version of this article

Iron-Nucleated Folding of a Metalloprotein in High Urea: Resolution of Metal Binding and Protein Folding Events

“…Using these protocols, the wt and C9S variant Rds were isolated as holo-iron proteins, C42S as partially iron loaded, and C39S and C6S as apoproteins. Metal contents were quantitated by inductively coupled plasma-atomic emission spectrometry at the University of Georgia Chemical Analysis Facility and by mass spectrometry (12). ApoRds used for the stopped-flow studies were verified to be metal free by these methods.…”

“…Due to their small size, well-characterized structural and spectroscopic properties, and their extraordinary resistance to denaturation and metal release, numerous investigations of thermal and chaotrope-induced unfolding of and metal loss from Rd have been reported (3–11). Our recent study (12), however, was the first to investigate the opposite process, namely, the polypeptide folding pathway of Rds and the influence of metal ion incorporation on this pathway.…”

“…In the case of the small copper-binding protein, azurin, dilution of the chaotrope-denatured apoprotein with Cu 2+ solutions led to much faster rates of recovery of the spectroscopically native copper site than when adding Cu 2+ to non-denatured apoprotein (14). We encountered a somewhat different metal incorporation/folding scenario for Rd (12). Dilution of chaotrope-denatured apoRd with iron-containing buffer resulted in only a minor portion of native, iron-containing Rd, the major portion consisting of a structured apoRd which was unable to incorporate iron.…”

The Key to the Extraordinary Thermal Stability of P. furiosus Holo-Rubredoxin: Iron Binding-Guided Packing of a Core Aromatic Cluster Responsible for High Kinetic Stability of the Native Structure

“…However, certain exceptions have been reported [5]–[7]. One relates to Pyrococcus furiosus rubredoxin (PfRd), an iron-sulphur cluster-containing protein reported to refold both in the presence [7], and absence of iron [8], [9].…”

“…However, certain exceptions have been reported [5]–[7]. One relates to Pyrococcus furiosus rubredoxin (PfRd), an iron-sulphur cluster-containing protein reported to refold both in the presence [7], and absence of iron [8], [9]. PfRd contains a single inorganic iron atom bound in tetrahedral geometry to the sulfur atoms of four cysteine residues in its sequence [numbered Cys5, Cys8, Cys38 and Cys41] through non-covalent coordinate bonds [10], [11].…”