CD AND MCD SPECTROSCOPIC STUDIES OF THE TWO DPS MINI-FERRITIN PROTEINS FROM B. ANTHRACIS: ROLE OF O2 AND H2O2 SUBSTRATES IN REACTIVITY OF THE DI-IRON CATALYTIC CENTERS

摘要

DNA Protection during Starvation (Dps) proteins are mini-ferritins found in bacteria and archaea that provide protection from uncontrolled Fe(II)/O radical chemistry; thus the catalytic sites are targets for antibiotics against pathogens, such as anthrax. Ferritin protein cages synthesize ferric oxymineral from Fe(II) and O2/H2O2, which accumulates in the large central cavity; for Dps, H2O2, is the more common Fe(II) oxidant contrasting with eukaryotic maxi-ferritins that often prefer dioxygen. To better understand the differences in the catalytic sites of maxi versus mini-ferritins, we used a combination of NIR circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field MCD (VTVH MCD) to study Fe(II) binding to the catalytic sites of the two B. anthracis mini-ferritins; one in which two Fe(II) react with O2 exclusively (Dps1) and a second in which both O2 or H2O2 can react with two Fe(II) (Dps2). Both result in the formation of iron oxy-biomineral. The data show: a single 5 or 6-coordinate Fe(II) in the absence of oxidant; Fe(II) binding to Dps2 is 30 × more stable than Dps1; and the lower limit of Kd for binding a second Fe(II), in the absence of oxidant, is 2–3 orders of magnitude weaker than for the binding of the single Fe(II). The data fit an equilibrium model where binding of oxidant facilitates formation of the catalytic site, in sharp contrast to eukaryotic M-ferritins where the binuclear Fe(II) centers are preformed before binding of O2. The two different binding sequences illustrate the mechanistic range possible for catalytic sites of the family of ferritins.