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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
2R37GM020194-20
Application #
3484350
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1975-06-01
Project End
1997-06-30
Budget Start
1992-07-01
Budget End
1993-06-30
Support Year
20
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of New Hampshire
Department
Type
Schools of Engineering
DUNS #
111089470
City
Durham
State
NH
Country
United States
Zip Code
03824
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Steere, Ashley N; Chasteen, N Dennis; Miller, Brendan F et al. (2012) Structure-based mutagenesis reveals critical residues in the transferrin receptor participating in the mechanism of pH-induced release of iron from human serum transferrin. Biochemistry 51:2113-21
Steere, Ashley N; Miller, Brendan F; Roberts, Samantha E et al. (2012) Ionic residues of human serum transferrin affect binding to the transferrin receptor and iron release. Biochemistry 51:686-94
Steere, Ashley N; Byrne, Shaina L; Chasteen, N Dennis et al. (2010) Evidence that His349 acts as a pH-inducible switch to accelerate receptor-mediated iron release from the C-lobe of human transferrin. J Biol Inorg Chem 15:1341-52
Steere, Ashley N; Roberts, Samantha E; Byrne, Shaina L et al. (2010) Properties of a homogeneous C-lobe prepared by introduction of a TEV cleavage site between the lobes of human transferrin. Protein Expr Purif 72:32-41
Byrne, Shaina L; Chasteen, N Dennis; Steere, Ashley N et al. (2010) The unique kinetics of iron release from transferrin: the role of receptor, lobe-lobe interactions, and salt at endosomal pH. J Mol Biol 396:130-40
Byrne, Shaina L; Steere, Ashley N; Chasteen, N Dennis et al. (2010) Identification of a kinetically significant anion binding (KISAB) site in the N-lobe of human serum transferrin. Biochemistry 49:4200-7
Mason, Anne B; Byrne, Shaina L; Everse, Stephen J et al. (2009) A loop in the N-lobe of human serum transferrin is critical for binding to the transferrin receptor as revealed by mutagenesis, isothermal titration calorimetry, and epitope mapping. J Mol Recognit 22:521-9
Mason, Anne B; Halbrooks, Peter J; James, Nicholas G et al. (2009) Structural and functional consequences of the substitution of glycine 65 with arginine in the N-lobe of human transferrin. Biochemistry 48:1945-53
Bou-Abdallah, Fadi; Biasiotto, Giorgio; Arosio, Paolo et al. (2004) The putative ""nucleation site"" in human H-chain ferritin is not required for mineralization of the iron core. Biochemistry 43:4332-7

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