CD and MCD spectroscopic studies of the two Dps miniferritin proteins from Bacillus anthracis: role of O2 and H2O2 substrates in reactivity of the diiron catalytic centers

Abstract

DNA protection during starvation (Dps) proteins are miniferritins 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 O(2)/H(2)O(2), which accumulates in the large central cavity; for Dps, H(2)O(2) is the more common Fe(II) oxidant contrasting with eukaryotic maxiferritins that often prefer dioxygen. To better understand the differences in the catalytic sites of maxi- versus miniferritins, 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 Bacillus anthracis miniferritins: one in which two Fe(II) react with O(2) exclusively (Dps1) and a second in which both O(2) or H(2)O(2) can react with two Fe(II) (Dps2). Both result in the formation of iron oxybiomineral. 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 K(D) 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 O(2). The two different binding sequences illustrate the mechanistic range possible for catalytic sites of the family of ferritins.

Figure 1

Representation of intersubunit active sites in (A) Dps1 and (B) Dps 2, obtained from crystal structures 1JI5 and 1JIG, respectively. All protein derived ligands are labeled and water is represented in red. In each structure the water that could occupy the possible second Fe(II) binding site is labeled. In Dps1 this water is 3.8 Å from the first Fe(II) site, and 4.1 Å away in Dps2

Figure 2

Anaerobic titration of Dps1 with Fe(II) in (A) CD and (B) MCD. (C) Binding curve with representative intensity for transition at 8300 cm⁻¹ plotted against the concentration of Fe(II) added. Fit parameters are given in Table 1.

Figure 3

(A) 278 K CD, 1.7 K CD, and 1.7 K, 7 Tesla MCD spectra of Dps1 with contribution of excess Fe(II) subtracted. Simultaneous peak fitting of these three spectra results in two transitions (gray): at 7800 and 10,870 cm⁻¹ (B) Overlay of 278 K CD (black), 2 K CD (red), and 2K, 7 Tesla MCD (green) taken of Dps2. These spectra are best fit with transitions at 8300 and 10300 cm⁻¹

Figure 4

Anaerobic titration of Dps2 with Fe(II) in (A) CD and (B) MCD. (C) MCD spectra of Fe(II) in a MOPS buffer solution (black), Dps2 with excess Fe(II) (red), and the subtraction spectrum of Dps2 minus Fe(II) in MOPS solution scaled to the appropriate concentration

Figure 5

Fits to VTVH MCD data collected on Dps1 at 7185 and 11000 cm⁻¹, using equation 2.

Figure 6

Fits to VTVH MCD data collected on Dps2 at 7800 and 11000 cm⁻¹ using equation 2

Figure 7

[P_f], [P₁], [P₂], & [Fe_f] are plotted as a function of log([Fe_T] / [P_T]) from the numerical solution to Equations 3 through 6. Total protein concentration is fixed at 240 µM while total Fe(II) concentration is varied from 0 to 1 M (~4200 Fe²⁺/Dps). K_d1 is fixed at 77 µM and K_d2 is fixed at 40,000 µM. The black line is the concentration of unbound Fe(II) in solution, red is the concentration of empty active sites in the Dps monomers, green is the concentration of mononuclear Fe(II) sites, and blue is the concentration of binuclear Fe(II) sites formed.

Figure 8

The apparent initial rates of Fe²⁺ oxidation scaled to overlay with the calculated concentration of binuclear Fe(II) active sites occupied at a given Fe²⁺/Dps ratio. A. Dps 1: The red line is the concentration of binuclear Fe(II) active sites occupied at a given Fe²⁺/Dps1 ratio calculated from equations 3 through 6 when K_D1 is fixed at 77 µM, K_D2 is fixed at 40,000 µM, and protein concentration is 240 µM. The blue line is the concentration of binuclear Fe(II) active sites occupied when K_D1 is fixed at 77 µM, and K_D2 is lowered to 15,000 µM. B. Dps2: The red line is the concentration of binuclear Fe(II) active sites occupied when K_D1 is fixed at 2.1 µM, K_D2 is fixed at 40,000 µM, and protein concentration is 240 µM. The blue line is the concentration of binuclear Fe(II) active sites occupied when K_D1 is fixed at 2.1 µM, and K_D2 is lowered to 15,000 µM.