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Fe(2+) substrate transport through ferritin protein cage ion channels influences enzyme activity and biomineralization.

ABSTRACT: Ferritins, complex protein nanocages, form internal iron-oxy minerals (Fe2O3·H2O), by moving cytoplasmic Fe(2+) through intracage ion channels to cage-embedded enzyme (2Fe(2+)/O2 oxidoreductase) sites where ferritin biomineralization is initiated. The products of ferritin enzyme activity are diferric oxy complexes that are mineral precursors. Conserved, carboxylate amino acid side chains of D127 from each of three cage subunits project into ferritin ion channels near the interior ion channel exits and, thus, could direct Fe(2+) movement to the internal enzyme sites. Ferritin D127E was designed and analyzed to probe properties of ion channel size and carboxylate crowding near the internal ion channel opening. Glu side chains are chemically equivalent to, but longer by one -CH2 than Asp, side chains. Ferritin D127E assembled into normal protein cages, but diferric peroxo formation (enzyme activity) was not observed, when measured at 650 nm (DFP ? max). The caged biomineral formation, measured at 350 nm in the middle of the broad, nonspecific Fe(3+)-O absorption band, was slower. Structural differences (protein X-ray crystallography), between ion channels in wild type and ferritin D127E, which correlate with the inhibition of ferritin D127E enzyme activity include: (1) narrower interior ion channel openings/pores; (2) increased numbers of ion channel protein-metal binding sites, and (3) a change in ion channel electrostatics due to carboxylate crowding. The contributions of ion channel size and structure to ferritin activity reflect metal ion transport in ion channels are precisely regulated both in ferritin protein nanocages and membranes of living cells.

SUBMITTER: Behera RK

PROVIDER: S-EPMC4634868 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Fe(2+) substrate transport through ferritin protein cage ion channels influences enzyme activity and biomineralization.

Behera Rabindra K RK Torres Rodrigo R Tosha Takehiko T Bradley Justin M JM Goulding Celia W CW Theil Elizabeth C EC

Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry 20150723 6

Ferritins, complex protein nanocages, form internal iron-oxy minerals (Fe2O3·H2O), by moving cytoplasmic Fe(2+) through intracage ion channels to cage-embedded enzyme (2Fe(2+)/O2 oxidoreductase) sites where ferritin biomineralization is initiated. The products of ferritin enzyme activity are diferric oxy complexes that are mineral precursors. Conserved, carboxylate amino acid side chains of D127 from each of three cage subunits project into ferritin ion channels near the interior ion channel exi ...[more]

PMID: 26202907

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Project description:Ferritins, a complex, mineralized, protein nanocage family essential for life, provide iron concentrates and oxidant protection. Protein-based ion channels and Fe(II)/O(2) catalysis initiate conversion of thousands of Fe atoms to caged, ferritin Fe(2)O(3)·H(2)O minerals. The ion channels consist of six helical segments, contributed by 3 of 12 or 24 polypeptide subunits, around the 3-fold cage axes. The channel structure guides entering Fe(II) ions toward multiple, catalytic, diiron sites buried inside ferritin protein helices, ~20 Å away from channel internal exits. The catalytic product, Fe(III)-O(H)-Fe(III), is a mineral precursor; mineral nucleation begins inside the protein cage with mineral growth in the central protein cavity (5-8 nm diameter). Amino acid substitutions that changed ionic or hydrophobic channel interactions R72D, D122R, and L134P increased ion channel structural disorder (protein crystallographic analyses) and increased Fe(II) exit [chelated Fe(II) after ferric mineral reduction/dissolution]. Since substitutions of some channel carboxylate residues diminished ferritin catalysis with no effect on Fe(II) exit, such as E130A and D127A, we investigated catalysis in ferritins with altered Fe(II) exit, R72D, D122R and L134P. The results indicate that simply changing the ionic properties of the channels, as in the R72D variant, need not change the forward catalytic rate. However, both D122R and L134P, which had dramatic effects on ferritin catalysis, also caused larger effects on channel structure and order, contrasting with R72D. All three amino acid substitutions, however, decreased the stability of the catalytic intermediate, diferric peroxo, even though overall ferritin cage structure is very stable, resisting 80 °C and 6 M urea. The localized structural changes in ferritin subdomains that affect ferritin function over long distances illustrate new properties of the protein cage in natural ferritin function and for applied ferritin uses.

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Fe(2+) substrate transport through ferritin protein cage ion channels influences enzyme activity and biomineralization.

Publications

Fe(2+) substrate transport through ferritin protein cage ion channels influences enzyme activity and biomineralization.

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