Facilitated Diffusion of Iron(II) and Dioxygen Substrates into Human H-Chain Ferritin. A Fluorescence and Absorbance Study Employing the Ferroxidase Center Substitution Y34W

Ferritin is a widespread iron mineralizing and detoxification protein that stores iron as a hydrous ferric oxide mineral core within a shell-like structure of 4/3/2 octahedral symmetry. Iron mineralization is initiated at dinuclear ferroxidase centers inside the protein where Fe2+ and O2 meet and react to form a μ-1,2-peroxodiferric intermediate that subsequently decays to form μ-oxo dimeric and oligomeric iron(III) species and ultimately the mineral core. Several types of channels penetrate the protein shell and are possible pathways for the diffusion of iron and dioxygen to the ferroxidase centers. In the present study, UV/visible and fluorescence stopped-flow spectrophotometries were used to determine the kinetics and pathways of Fe2+ diffusion into the protein shell, its binding at the ferroxidase center and its subsequent oxidation by O2. Three fluorescence variants of human H-chain ferritin were prepared in which Trp34 was introduced near the ferroxidase center. They included a control variant no. 1 (W93F/Y34W), a "1-fold" channel variant no. 2 (W93F/Y34W/Y29Q) and a 3-fold channel variant no. 3 (Y34W/W93F/D131I/E134F). Anaerobic rapid mixing of Fe2+ with apo-variant no. 1 quenched the fluorescence of Trp34 with a rate exhibiting saturation kinetics with respect to Fe2+ concentration, consistent with a process involving facilitated diffusion. A half-life of ∼3 ms for this process is attributed to the time for diffusion of Fe2+ across the protein shell to the ferroxidase center. No fluorescence quenching was observed with the 3-fold channel variant no. 3 or when Zn2+ was prebound in each of the eight 3-fold channels of variant no. 1, observations indicating that the hydrophilic channels are the only avenues for rapid Fe2+ access to the ferroxidase center. Substitution of Tyr29 with glutamine at the entrance of the "1-fold" hydrophobic channel had no effect on the rate of Fe2+ oxidation to form the μ-1,2-peroxodiferric complex (t1/2 ≈ 38 ms), a finding demonstrating that Tyr29 and, by inference, the "1-fold" channels do not facilitate O2 transport to the ferroxidase center, contrary to predictions of DFT and molecular dynamics calculations. O2 diffusion into ferritin occurs on a time scale that is fast relative to the millisecond kinetics of the stopped-flow experiment.