title: The ferroxidase reaction of ferritin reveals a diferric mu-1,2 bridging peroxide intermediate in common with other O2-activating non-heme diiron proteins.
abstract: Ferritins are ubiquitous proteins that concentrate, store, and detoxify intracellular iron through oxidation of Fe2+ (ferroxidation), followed by translocation and hydrolysis to form a large inorganic mineral core. A series of mutagenesis, kinetics, and spectroscopic studies of ferritin led to the proposal that the oxidation/translocation path involves a diiron protein site. Recent stopped-flow absorption and rapid freeze-quench Mössbauer studies have identified a single peroxodiferric species as the initial transient intermediate formed in recombinant frog M ferritin during rapid ferroxidation [Pereira, S. A., Small, W., Krebs, C., Tavares, P., Edmondson, D. E., Theil, E. C., and Huynh, B. H. (1998) Biochemistry 37, 9871-9876]. To further characterize this transient intermediate and to establish unambiguously the peroxodiferric assignment, rapid freeze-quenching was used to trap the initial intermediate for resonance Raman investigation. Discrete vibrational modes are observed for this intermediate, indicating a single chromophore in a homogeneous state, in agreement with the Mössbauer conclusions. The frequency at 851 cm-1 is assigned as nu(O-O) of the bound peroxide, and the pair of frequencies at 485 and 499 cm-1 is attributed, respectively, to nus and nuas of Fe-O2-Fe. Identification of the chromophore as a micro-1,2 bridged diferric peroxide is provided by the isotope sensitivity of these Raman bands. Similar peroxodiferric intermediates have been detected in a mutant of the R2 subunit of ribonucleotide reductase from Escherichia coli and chemically reduced Delta9 stearoyl-acyl carrier protein desaturase (Delta9D), but in contrast, the ferritin intermediate is trapped from the true reaction pathway of the native protein. Differences in the Raman signatures of these peroxide species are assigned to variations in Fe-O-O-Fe angles and may relate to whether the iron is retained in the catalytic center or released as an oxidized product.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland 97291-1000, USA. ploccoz@bmb.ogi.edu
authors.forename: P
authors.initials: P
authors.lastname: Moënne-Loccoz
authors.forename: C
authors.initials: C
authors.lastname: Krebs
authors.forename: K
authors.initials: K
authors.lastname: Herlihy
authors.forename: D E
authors.initials: DE
authors.lastname: Edmondson
authors.forename: E C
authors.initials: EC
authors.lastname: Theil
authors.forename: B H
authors.initials: BH
authors.lastname: Huynh
authors.forename: T M
authors.initials: TM
authors.lastname: Loehr
mesh_terms: D000818:Animals; D001052:Apoferritins / Q000737:chemistry; D002570:Ceruloplasmin / Q000737:chemistry*; D005290:Ferric Compounds / Q000737:chemistry*; D005293:Ferritins / Q000737:chemistry*; D007501:Iron / Q000737:chemistry*; D019159:Nonheme Iron Proteins / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry*; D010103:Oxygen Isotopes; D010545:Peroxides / Q000737:chemistry*; D011898:Ranidae; D013059:Spectrum Analysis, Raman; D013379:Substrate Specificity
chemical_list: D005290:Ferric Compounds; D019159:Nonheme Iron Proteins; D010103:Oxygen Isotopes; D010545:Peroxides; D005293:Ferritins; D001052:Apoferritins; D007501:Iron; D002570:Ceruloplasmin; D010100:Oxygen