The consequences of hydroxyl radical formation on the stoichiometry and kinetics of ferrous iron oxidation by human apoferritin

Despite previous detection of hydroxyl radical formation during iron deposi tion into ferritin, no reports exist in the literature concerning how it mi ght affect ferritin function. In the present study, hydroxyl radical format ion during Fe(II) oxidation by apoferritin was found to be contingent on th e "ferroxidase" activity (i.e., H subunit composition) exhibited by apoferr itin. Hydroxyl radical formation was found to affect both the stoichiometry and kinetics of Fe(II) oxidation by apoferritin. The stoichiometry of Fe(I I) oxidation by apoferritin in an unbuffered solution of 50 mM NaCl, pH 7.0 , was approximately 3.1 Fe(ll)/O-2 at all iron-to-protein ratios tested. Th e addition of HEPES as an alternate reactant for the hydroxyl radical resul ted in a stoichiometry of about 2 Fe(ll)/O-2 at all iron-to-protein ratios. HEPES functioned to protect apoferritin from oxidative modification, for i ts omission from reaction mixtures containing Fe(II) and apoferritin result ed in alterations to the ferritin consistent with oxidative damage. The kin etic parameters for the reaction of recombinant human H apoferritin with Fe (II) in HEPES buffer (100 mM) were: K-m = 60 muM, k(cat) = 10 s(-1), and k( cat)/K-m = 1.7 X 10(5) M-1 . s(-1). collectively, these results contradict the "crystal growth model" for iron deposition into ferritin and, while our data would seem to imply that the ferroxidase activity of ferritin is adeq uate in facilitating Fe(II) oxidation at all stages of iron deposition into ferritin, it is important to note that these data were obtained in vitro u sing nonphysiologic conditions. The possibility that these findings may hav e physiological significance is discussed. (C) 2001 Elsevier Science Inc.