Mechanism and kinetics of iron release from ferritin by dihydroflavins and dihydroflavin analogs

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Abstract
Dihydroflavins reductively release Fe rapidly and quantitatively from purified horse spleen or horse heart ferritin. The NAD(P)H:flavin oxidoreductase from Beneckea harveyi is used to generate a constant concentration of dihydroflavin permitting a continuous assay for complete Fe release. Sepharose-linked dihydroflavins are not competent to release ferritin Fe, demonstrating that the dihydroflavin must pass through the channels of the protein shell prior to Fe reduction. Several experiments fail to show any specific flavin binding site, though dihydroflavins do display saturation kinetics with very high apparent Km. The rates of Fe release by a number of dihydroflavin analogs show that the electron transfer is significantly rate determining in Fe release by dihydroriboflavin, while diffusion of the dihydroflavin through the protein channel is slow in the release of Fe by dihydroFMN. The rate of Fe release is also dependent on the initial content of Fe, having a maximum at 1200 Fe atoms/ferritin.