Iron is an important nutrient, required in almost every aspect of cellular function. However, at physiological pH and under oxidizing condition, it is not very soluble. How living organisms sequester iron for cellular utilization is therefore a fundamental question of vital importance. Also, in the presence Of 02, free Fe2+ ions are extremely toxic, capable of generating hydrogen peroxide, superoxide, and other reactive oxygen species that can attack and destroy important cellular molecules. Ferritin is unique in the sense that it performs dual functions of iron detoxification, by oxidizing the Fe2+ ions in solution, and iron sequestration, by storing the oxidized Fe3+ ions in its inner protein cavity in the form of ferrihydrite mineral. However, despite the importance of ferritin functions and decades of research efforts, the mechanism by which ferritin catalyzes the Fe2+ oxidation (ferroxidation) and directs the oxidized products to form the mineral core (mineralization) is still poorly understood. This is partly due to the complexity of the ferritin molecule and partly due to the fact that the methods used in previous studies were either indirect or lacked the required spectroscopic resolution to monitor the complex reaction catalyzed by ferritin. In this application, we propose to employ M?ssbauer spectroscopy in conjunction with the rapid freeze-rapid quench kinetic technique to investigate the mechanism of ferritin ferroxidation and mineralization. Three different recombinant ferritins, the frog H and M ferritins, and the E. coli bacterioferritin, are to be examined. Results obtained from our preliminary studies demonstrate that this combined kinetic/spectroscopic approach provides the necessary time resolution for obtaining kinetic information and the required spectroscopic resolution for distinguishing, quantifying and characterizing the multiple Fe species generated during the ferroxidation and mineralization processes. Other complementary spectroscopies, such as EPR, ENDOR, EXAFS, and resonance Raman will also be employed to obtain further structural information on these reaction intermediates. Site-specific mutants will be engineered, produced and subjected to kinetic/spectroscopic investigations for the purpose of defining the ferroxidase site, the Fe transport pathways, and the functional roles of certain key residues. A series of double-mixing rapid freeze-quench Mossbauer investigations using 57Fe and 56Fe isotopes are particularly designed to address questions concerning the dynamics of the ferritin function. Detailed mechanistic insights into the processes involved in ferritin ferroxidase reaction and mineral core formation are expected to emerge from these proposed studies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058778-03
Application #
6343059
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Preusch, Peter C
Project Start
1999-01-01
Project End
2002-12-31
Budget Start
2001-01-01
Budget End
2001-12-31
Support Year
3
Fiscal Year
2001
Total Cost
$183,998
Indirect Cost
Name
Emory University
Department
Physics
Type
Schools of Arts and Sciences
DUNS #
042250712
City
Atlanta
State
GA
Country
United States
Zip Code
30322
Yun, Danny; Saleh, Lana; Garcia-Serres, Ricardo et al. (2007) Addition of oxygen to the diiron(II/II) cluster is the slowest step in formation of the tyrosyl radical in the W103Y variant of ribonucleotide reductase protein R2 from mouse. Biochemistry 46:13067-73
Baldwin, Jeffrey; Krebs, Carsten; Saleh, Lana et al. (2003) Structural characterization of the peroxodiiron(III) intermediate generated during oxygen activation by the W48A/D84E variant of ribonucleotide reductase protein R2 from Escherichia coli. Biochemistry 42:13269-79
Silaghi-Dumitrescu, Radu; Coulter, Eric D; Das, Amaresh et al. (2003) A flavodiiron protein and high molecular weight rubredoxin from Moorella thermoacetica with nitric oxide reductase activity. Biochemistry 42:2806-15
Jameson, Guy N L; Jin, Weili; Krebs, Carsten et al. (2002) Stoichiometric production of hydrogen peroxide and parallel formation of ferric multimers through decay of the diferric-peroxo complex, the first detectable intermediate in ferritin mineralization. Biochemistry 41:13435-43
Yun, Danny; Krebs, Carsten; Gupta, Govind P et al. (2002) Facile electron transfer during formation of cluster X and kinetic competence of X for tyrosyl radical production in protein R2 of ribonucleotide reductase from mouse. Biochemistry 41:981-90
Krebs, Carsten; Bollinger Jr, J Martin; Theil, Elizabeth C et al. (2002) Exchange coupling constant J of peroxodiferric reaction intermediates determined by Mossbauer spectroscopy. J Biol Inorg Chem 7:863-9
Pereira, A S; Tavares, P; Moura, I et al. (2001) Mossbauer characterization of the iron-sulfur clusters in Desulfovibrio vulgaris hydrogenase. J Am Chem Soc 123:2771-82
Ghiladi, R A; Hatwell, K R; Karlin, K D et al. (2001) Dioxygen reactivity of mononuclear heme and copper components yielding a high-spin heme-peroxo-cu complex. J Am Chem Soc 123:6183-4
Hwang, J; Krebs, C; Huynh, B H et al. (2000) A short Fe-Fe distance in peroxodiferric ferritin: control of Fe substrate versus cofactor decay? Science 287:122-5
Jovanovic, T; Ascenso, C; Hazlett, K R et al. (2000) Neelaredoxin, an iron-binding protein from the syphilis spirochete, Treponema pallidum, is a superoxide reductase. J Biol Chem 275:28439-48