Mechanism and kinetics of iron release from ferritin by dihydroflavins and dihydroflavin analogues.

Dihydroflavins reductively release iron 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 iron release. Sepharose-linked dihydroflavins are not competent to release ferritin iron, demonstrating that the dihydroflavin must pass through the channels of the protein shell prior to iron reduction. Several experiments fail to show any specific flavin binding site, though dihydroflavins do display saturation kinetics with very high apparent Km's. The rates of iron release by a number of dihydroflavin analogues show that the electron transfer is significantly rate determining in iron release by dihydroriboflavin, while diffusion of the dihydroflavin through the protein channel is slow in the release of iron by dihydroFMN. The rate of iron release is also dependent on the initial content of iron, having a maximum at 1200 iron atoms per ferritin.