Molecular diffusion into horse spleen ferritin: a nitroxide radical spin probe study

Abstract

Electron paramagnetic resonance spectroscopy and gel permeation chromatography were employed to study the molecular diffusion of a number of small nitroxide spin probes (approximately 7-9 A diameter) into the central cavity of the iron-storage protein ferritin. Charge and polarity of these radicals play a critical role in the diffusion process. The negatively charged radical 4-carboxy-2,2,6,6-tetramethylpiperidine-N-oxyl (4-carboxy-TEMPO) does not penetrate the cavity whereas the positively charged 4-amino-TEMPO and 3-(aminomethyl)-proxyl radical and polar 4-hydroxy-TEMPO radical do. Unlike the others, the apolar TEMPO radical does not enter the cavity but instead binds to ferritin, presumably at a hydrophobic region of the protein. The kinetic data indicate that diffusion is not purely passive, the driving force coming not only from the concentration gradient between the inside and outside of the protein but also from charge interactions between the diffusant and the protein. A model for diffusion is derived that describes the observed kinetics. First-order half-lives for diffusion into the protein of 21-26 min are observed, suggesting that reductant molecules with diameters considerably larger than approximately 9 A would probably enter the protein cavity too slowly to mobilize iron efficiently by direct interaction with the mineral core.

A New Role for Heme, Facilitating Release of Iron from the Bacterioferritin Iron Biomineral

“…The literature on the ability of small molecules to penetrate the coat of ferritin protein is not clear (45), but it is possible that chelators may be able to access the BFR cavity and in doing so may influence the rate at which iron exits the protein. However, if the reduction step is rate-limiting, why should this affect the overall rate of release?…”

A Protein-Based Ferritin Bio-Nanobattery

“…In its biological role as an iron storage protein, the surrounding protein shell protects the iron mineral core from reaction with the exterior solution and the channels function as pores through this "membrane shell" and serve the unique role of ion transport. Large or negatively charged species are excluded, but small ions and molecules readily enter and leave the ferritin interior through these channels [11,12]. The assembled structure of ferritins is remarkably stable to biological extremes of temperature (80…”

“…The hydrophylic 3-fold channels are used for ion transfer into and out of the ferritin but the role of the hydrophobic 4-fold channels remain unclear. However, that ions and molecules enter and leave through these channels is well documented [12]. Trapping large organic dye molecules and ferricyanide in the ferritin interior established [11] that their large size precluded their release.…”

Rapid Mapping of Active Site of KSI by Paramagnetic NMR

“…15 In contrast, HyTEMPO is approximately 6.5 × 8.7 Å. 28 Thus, HyTEMPO can easily enter in hydrophobic cavity due to the slightly large size of hydrophobic cavity. These characteristics of HyTEMPO can enable rapid mapping of binding site between the substrate (or analog) and KSI.…”