FURTHER CHARACTERIZATION OF THE REDOX AND SPECTROSCOPIC PROPERTIES OF AZOTOBACTER-VINELANDII FERRITIN

Bacterial ferritin from Azotobacter vinelandii (AVBF) has many properties in common with and a number of properties distinct from the more thoroughly studied animal ferritins. The most notable differences are the high phosphate content of the mineral core and the presence of heme (12 per AVBF) in AVBF. In both ferritin types, redox reactions are essential to the iron release and deposition function of the ferritins. The heme reduction potential in apo AVBF is pH independent as are both the heme and core reduction potential in holo AVBF. pH measurements confirm the pH independence for heme reduction in apo AVBF; however, they establish the conflicting result that 1.7 +/- 0.2 protons per iron atom are taken up during core reduction. These results are interpreted as a two-step reduction process consisting of a pH independent reduction of heme in holo AVBF followed by a pH dependent reduction of the mineral core. Detailed spectroscopic studies have been undertaken to determine if heme-core interactions are detectable during the redox reactions of AVBF. Optical spectroscopy of the heme groups in apo AVBF demonstrate that all twelve are identical and undergo uniform and rapid reduction. EPR spectroscopy establishes the presence of both low-symmetry, g = 4.3, Fe3+ from the mineral core and low-spin heme with g values of 2.87, 2.32, and 1.46 in holo and identical g values for the low-spin heme in apo AVBF. EPR integration of the heme groups in both apo and holo gave values of 13.2 + 1.3 heme spins per AVBF at 4.2, 10, 25, 35, and 45 K. No heme perturbations were detected in holo or apo AVBF by Resonance Raman and circular dichroism spectroscopy. Both reduced and oxidized apo AVBF gave normal fluorescence emission at 330-340 nm when excited at 279 nm. These spectroscopic redox, and reactivity results provide more detailed properties of AVBF for comparison with other bacterial and animal ferritins.