DINUCLEAR CENTER OF FERRITIN - STUDIES OF IRON-BINDING AND OXIDATION SHOW DIFFERENCES IN THE 2 IRON SITES

The ferroxidase activity of human ferritin has previously been associa ted with a diiron site situated centrally within the four-helix bundle of H-type chains (HuHF), However, direct information about the site o f Fe(II) binding has been lacking, and events between Fe(II) binding a nd its oxidation have not previously been studied. A sequential stoppe d-flow assay has now been developed to enable the dissection of bindin g and oxidation. It depends on the ability of 1,10-phenanthroline to c omplex protein-bound Fe(II) and to distinguish it from the more immedi ately available free Fe(II). This approach, aided by the use of site-d irected variants, indicates that in HuHF and the non-heme ferritin of Escherichia coli the first 48 Fe(II) atoms/molecule added are bound an d oxidized at the dinuclear centers. At a constant iron concentration, the rate of Fe(ll) oxidation was maximal for additions of 2 Fe(II) at oms/subunit, consistent with a two-electron oxidation of the Fe(II) pa ir, Although, at low Fe(II)/protein ratios, no cooperativity in Fe(II) binding was observed: a preferred order of binding was deduced [Fe(II ) binding first at site A and then at site B], Binding of Fe(II) at bo th sites was essential for fast oxidation. Modification of site A liga nds resulted in slow iron binding and slow oxidation, Modification of site B did not prevent Fe(II) binding at site A but greatly reduced it s oxidation rate. These differences may mean that dioxygen is initiall y bound to Fe(II) at site B.