Fe2+ 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 Fe2+ through intracage ion channels to cage-embedded enzyme (2Fe2+/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 Fe2+ 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 650nm (DFP λmax). The caged biomineral formation, measured at 350 nm in the middle of the broad, nonspecific Fe3+-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.

Self-assembly Is Prerequisite for Catalysis of Fe(II) Oxidation by Catalytically Active Subunits of Ferritin

“…A similar conclusion was derived in a more recent study in which a single amino acid substitution in the 3-fold symmetry axis of BfMF, i.e. D127E, abolished the rate of Fe(II) oxidation in the ferroxidase center ( 51 ). However, in both studies it has not been checked whether in BfMF mutation of the residues at 3-fold symmetry axis affects assembly of the protein, possibly resulting in inability of the ferroxidase center to properly bind the Fe(II) ions and thus to abolishment of Fe(II) oxidation in the ferroxidase center as here observed in PfFtn.…”

“…However, in both studies it has not been checked whether in BfMF mutation of the residues at 3-fold symmetry axis affects assembly of the protein, possibly resulting in inability of the ferroxidase center to properly bind the Fe(II) ions and thus to abolishment of Fe(II) oxidation in the ferroxidase center as here observed in PfFtn. In fact for the D127E mutant of BfMF, x-ray crystallography showed only monomers ( 51 ) and not the self-assembled 24-meric structure reported for wild-type BfMF ( 52 ). In a separate study using x-ray crystallography, the molecular structure of WT-BfMF was solved after aerobic and anaerobic incubation with Fe(II) for different time intervals ( 45 ).…”

“…Instead R117A mutation should have affected Fe(II) binding and oxidation at the ferroxidase center and lower thermostability of R117A-PfFtn compared with WT-PfFtn by abolishing the self-assembly. These results appear to be not in favor of a model in which Fe(II) needs to enter the internal cavity of protein and from there reaches the ferroxidase center via individual metal ion binding sites in the internal surface of ferritin including site C. In this light, we re-evaluated three recent studies based on which a route of Fe(II) to the ferroxidase center through the internal cavity and via the conserved site C was defined ( 29 , 45 , 51 ).…”

Routes of iron entry into, and exit from, the catalytic ferroxidase sites of the prokaryotic ferritin SynFtn

“…S1 †), and both crystallographic data and disruption of this channel by site directed mutagenesis demonstrate that this is the route of iron entry into these proteins. 5,10,11 Specifically, two conserved carboxylate residues (Asp127 and Glu130, Frog M numbering) generate two symmetry-related binding sites for hexaquo-Fe 2+ within each channel.…”

Genome-wide comparison of ferritin family from Archaea, Bacteria, Eukarya, and Viruses: its distribution, characteristic motif, and phylogenetic relationship

“…However, it is toxic of excess iron in the cellular, and free Fe 2+ donates electrons to produce hydroxyl radicals via the Fenton reaction (Fe 2+ +H 2 O 2 →Fe 3+ +OH − +HO . (Behera et al 2015;Harrison and Arosio 1996). Free hydroxyl radicals can catalyze the oxidative damage of biomolecules; nature's answer to this dual problem of availability and toxicity is the ferritin family of iron storage proteins (Bradley et al 2014;Torti and Torti 2002), which are ancient sizeable multi-subunit proteins found in most cell types of humans and other vertebrates, invertebrates, plants, fungi, bacteria, and archaea (Harrison 1986;Ragland et al 1990;Theil 1987).…”