Stoichiometric production of hydrogen peroxide and parallel formation of ferric multimers through decay of the diferric-peroxo complex, the first detectable intermediate in ferritin mineralization.
The catalytic step that initiates formation of the ferric oxy-hydroxide mineral core in the central cavity of H-type ferritin involves rapid oxidation of ferrous ion by molecular oxygen (ferroxidase reaction) at a binuclear site (ferroxidase site) found in each of the 24 subunits. Previous investigators have shown that the first detectable reaction intermediate of the ferroxidase reaction is a diferric-peroxo intermediate, F(peroxo), formed within 25 ms, which then leads to the release of H(2)O(2) and formation of ferric mineral precursors. The stoichiometric relationship between F(peroxo), H(2)O(2), and ferric mineral precursors, crucial to defining the reaction pathway and mechanism, has now been determined. To this end, a horseradish peroxidase-catalyzed spectrophotometric method was used as an assay for H(2)O(2). By rapidly mixing apo M ferritin from frog, Fe(2+), and O(2) and allowing the reaction to proceed for 70 ms when F(peroxo) has reached its maximum accumulation, followed by spraying the reaction mixture into the H(2)O(2) assay solution, we were able to quantitatively determine the amount of H(2)O(2) produced during the decay of F(peroxo). The correlation between the amount of H(2)O(2) released with the amount of F(peroxo) accumulated at 70 ms determined by Mössbauer spectroscopy showed that F(peroxo) decays into H(2)O(2) with a stoichiometry of 1 F(peroxo):H(2)O(2). When the decay of F(peroxo) was monitored by rapid freeze-quench Mössbauer spectroscopy, multiple diferric mu-oxo/mu-hydroxo complexes and small polynuclear ferric clusters were found to form at rate constants identical to the decay rate of F(peroxo). This observed parallel formation of multiple products (H(2)O(2), diferric complexes, and small polynuclear clusters) from the decay of a single precursor (F(peroxo)) provides useful mechanistic insights into ferritin mineralization and demonstrates a flexible ferroxidase site.
Jameson
Guy N L
GN
Department of Physics, Emory University, Atlanta, Georgia 30322, USA.
Jin
Weili
W
Krebs
Carsten
C
Perreira
Alice S
AS
Tavares
Pedro
P
Liu
Xiaofeng
X
Theil
Elizabeth C
EC
Huynh
Boi Hanh
BH
D000818:Animals; D001052:Apoferritins / Q000737:chemistry / Q000378:metabolism; D002570:Ceruloplasmin / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D005290:Ferric Compounds / Q000737:chemistry / Q000378:metabolism*; D005293:Ferritins / Q000737:chemistry / Q000378:metabolism*; D005615:Freezing; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism*; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D046911:Macromolecular Substances; D011892:Rana catesbeiana; D015204:Spectroscopy, Mossbauer
D005290:Ferric Compounds; D046911:Macromolecular Substances; D005293:Ferritins; D001052:Apoferritins; D006861:Hydrogen Peroxide; D007501:Iron; D002570:Ceruloplasmin