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 approximate to10(14) times above the ferric K-s. 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 (F-ox) sites, complicated by multiple types of iron-ferritin interactions, is now achieved with chimeric proteins where putative F-ox 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 F-ox intermediates (A at 650 nm) monitored activity. Diferric peroxo formation in chimeric and WT proteins had similar K-app 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 kat 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.