IRON DEPOSITION AND MOBILIZATION IN FERRITIN

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From microorganisms to man, ferritin plays a central role in the biological management of iron. Within the cell, iron is reversibly stored in ferritin in the form of a hydrous ferric oxide mineral core inside the protein. Ferritin consists of 24 subunits of two types, H (heavy) and L (light) of apparent molecular weights 21,000 and 19,000 g/mole, respectively, assembled to form a shell of molecular weight c.a. 480,000 g/mol. The two subunits have been suggested to have different roles in facilitating core formation in ferritin. A ferroxidase site which catalyzes the oxidation of iron(II) by molecular oxygen has recently been discovered on the H-subunit. The overall goal of the proposed research is to elucidate the molecular mechanisms by which ferritin acquires and releases iron with special attention paid to the roles of the H and L subunits in these processes. The mechanisms will be investigated through a combination of EPR, ENDOR, ESEEM and Mossbauer spectroscopy, electrochemistry and kinetic studies of horse and sheep spleen ferritins as well as recombinant H and L human liver ferritins and site-directed mutants thereof. The various EPR observable iron and radical species formed during the initial stages of iron deposition will be characterized spectroscopically and kinetically and their locations within the protein structure determined using mutant proteins and proteins enriched in selected perdeutero amino acids. The role(s), if any, of the observed radicals, in iron oxidation and/or reduction or in oxidative damage to the protein will be examined. The kinetics of iron oxidation and hydrolysis will be studied by oxygen electrode oximetry and pH stat, respectively, and the rate laws for these processes determined. The time sequence of formation of intermediate mononuclear and binuclear iron and radical species will be established by rapid freeze-quench EPR spectroscopy. The functions of H and L subunits in facilitating iron oxidation and core nucleation will be probed by carrying out spectroscopic and kinetic studies of intermediate species observed with various site-directed mutants. The binding of biological reductants to ferritin and the reversibility of the redox chemistry of the iron core will be studied electrochemically. The extensive studies proposed should lead to a detailed understanding of how ferritin functions as a reversible iron storage protein and further our knowledge of the chemistry and biochemistry of iron biomineralization processes in general.