Ferritin reactions: Direct identification of the site for the diferric peroxide reaction intermediate

Ferritins managing iron–oxygen biochemistry in animals, plants, and microorganisms belong to the diiron carboxylate protein family and concentrate iron as ferric oxide ≈1014 times above the ferric Ks. Ferritin iron (up to 4,500 atoms), used for iron cofactors and heme, or to trap DNA-damaging oxidants in microorganisms, is concentrated in the protein nanocage cavity (5–8 nm) formed during assembly of polypeptide subunits, 24 in maxiferritins and 12 in miniferritins/DNA protection during starvation proteins. Direct identification of ferritin ferroxidase (Fox) sites, complicated by multiple types of iron–ferritin interactions, is now achieved with chimeric proteins where putative Fox site residues were introduced singly and cumulatively into an inactive host, an L maxiferritin. A dimagnesium ferritin cocrystal model guided site design and the diferric peroxo Fox intermediates (A at 650 nm) monitored activity. Diferric peroxo formation in chimeric and WT proteins had similar Kapp values and Hill coefficients. Catalytic activity required cooperative ferrous substrate binding to two sites A (E, EXXH) and B (E, QXXD). The weaker B sites in ferritin contrast with stronger B sites (E, EXXH) in diiron carboxylate oxygenases, explaining diferric oxo/hydroxo product release in ferritin vs. diiron cofactor retention in oxygenases. Codons for Q/H and D/E differ by single nucleotides, suggesting simple DNA mutations relate site B diiron substrate sites and diiron cofactor sites in proteins. The smaller kcat values in chimeras indicate the absence of second-shell residues important for ferritin substrate–product channeling that, when identified, will outline the entire iron path from ferritin pores through the Fox site to the mineral cavity.