A Kinetic Study of Iron Release from Azotobacter Vinelandii Bacterial Ferritin

The kinetics of iron release from Azotobacter vinelandii bacterial ferritin (AVBF) was measured by reduction of core iron with S2O4(2-) followed by chelation of Fe2+ with alpha, alpha-bipyridine (bipy). The rate was first order in AVBF and one half order in S2O4(2-), suggesting that SO2- is the active reductant formed by S2O4(2-) = 2SO2-. With zero-order conditions for dithionite and bipy, two consecutive first-order iron release reactions differing by a factor of about 14 were observed with rate constants of 0.0263 and 0.00184 sec-1, respectively, at 25 degrees C and pH 7.0. The faster reaction corresponded to the loss of 1433 iron atoms (91%) and the slower second reaction corresponded to loss of 145 (9%) of the original 1575 iron atoms present. The first reaction increased about twofold with pH variation between 6.5 and 8.0, whereas the second reaction was unchanged in the pH range 5.5-8. Both dramatically increased at pH 5.0. Methyl viologen increased the rate of both reactions about tenfold. The biphasic behavior for iron loss is interpreted as two different populations of iron atoms present in the core of AVBF, the first representing the bulk iron, and the second a group of unique iron atoms released last which may represent iron attached to the interior of the protein shell or iron associated with the heme groups. Kinetic stopped-flow measurements show that the heme is first reduced, followed by reduction of the core iron by reduced heme, suggesting an electron transfer role for heme in AVBF function.