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The kinetics for complete iron release showing biphasic behavior from pig spleen ferritin-Fe (PSFF) was measured by spectrophotometry. The native core within the PSFF shell consisted of 1682 hydroxide Fe3+ and 13 phosphate molecules. Inhibition kinetics for complete iron release was measure by differential spectrophotometry in the presence of phosphate; the process was clearly divided into two phases involving a first-order reaction at an increasing rate of 46.5 Fe3+/PSFF/min on the surface of the iron core and a zero-order reaction at a decreasing rate of 6.67 Fe3+/PSFF/min inside the core. The kinetic equation [C(PSFF-Fe3+)max - C(PSFF-Fe3+)t](1/2) = Tmax - Tt gives the transition time between the two rates and represents the complex kinetic characteristics. The rate was directly accelerated twofold by a mixed reducer of dithionite and ascorbic acid. These results suggest that the channel of the PSFF shell may carry out multiple functions for iron metabolism and storage and that the phosphate strongly affects the rate of iron release.

In bioelectrochemical studies, redox mediators such as methylene blue, natural red, and thionine are used to studying the redox characteristics of enzymes in the living cell. Here we show that nitrogenase activity in Azotobacter vinelandii is completely inhibited by oxidized methylene blue (MBo) when the concentration of this mediator in the medium is increased up to 72 microM. This activity in A. vinelandii is somewhat inhibited by a coenzyme, ascorbic acid (AA). However, the nitrogenase activity within the A. vinelandii cell is unchanged even for a high concentration of oxidized natural red (NRo) alone. Interestingly, these mediators and AA do not have the capacity to inhibit the H2 uptake activity of the hydrogenase in A. vinelandii. Average active rates of 66 nM H2 evolved/mg cell protein/min from the nitrogenase and 160 nM H2-uptake/mg cell protein/min from the hydrogenase in A. vinelandii are found in aid of the activities of the enzymes for H2 evolution and for H2 uptake are compared. The activities of both enzymes in A. vinelandii are strongly inhibited by thionine having high oxidative potential. Mechanisms of various mediators acting in vivo for both enzymes in A. vinelandii are discussed.