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 Fe(2+) and O(2) meet and react to form a mu-1,2-peroxodiferric intermediate that subsequently decays to form mu-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 Fe(2+) diffusion into the protein shell, its binding at the ferroxidase center and its subsequent oxidation by O(2). 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 Fe(2+) with apo-variant no. 1 quenched the fluorescence of Trp34 with a rate exhibiting saturation kinetics with respect to Fe(2+) concentration, consistent with a process involving facilitated diffusion. A half-life of approximately 3 ms for this process is attributed to the time for diffusion of Fe(2+) across the protein shell to the ferroxidase center. No fluorescence quenching was observed with the 3-fold channel variant no. 3 or when Zn(2+) 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 Fe(2+) 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 Fe(2+) oxidation to form the mu-1,2-peroxodiferric complex (t(1/2) approximately 38 ms), a finding demonstrating that Tyr29 and, by inference, the "1-fold" channels do not facilitate O(2) transport to the ferroxidase center, contrary to predictions of DFT and molecular dynamics calculations. O(2) diffusion into ferritin occurs on a time scale that is fast relative to the millisecond kinetics of the stopped-flow experiment.
Bou-Abdallah
Fadi
F
Department of Chemistry, State University of New York, Potsdam, New York 13676, USA.
Zhao
Guanghua
G
Biasiotto
Giorgio
G
Poli
Maura
M
Arosio
Paolo
P
Chasteen
N Dennis
ND
D001052:Apoferritins / Q000737:chemistry* / Q000378:metabolism; D002151:Calorimetry; D020134:Catalytic Domain; D002570:Ceruloplasmin / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D004058:Diffusion; D005296:Ferrous Compounds / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013056:Spectrophotometry, Ultraviolet
D005296:Ferrous Compounds; D001052:Apoferritins; D006861:Hydrogen Peroxide; D002570:Ceruloplasmin; D010100:Oxygen