D. Cope Norcross and Dr. Gerald D. Watt, Chemistry and Biochemistry
Iron is essential to all forms of life. Yet, free iron is toxic unless sequestered and regulated. Organisms use the protein ferritin to perform this function. Ferritin is found storing and releasing iron in almost all living organisms. Ferritin is a large, nearly spherical, hollow protein composed of twenty-four nearly identical subunits (Harrison, et. al., 1991; Theil, 1990). The structure of this protein has been characterized and is well known, yet relating this form to its function is still an active area of research. Although there have been many studies done and many models proposed, very little is known about how ferritin stores and releases iron.
Recently investigators have determined that the protein shell of ferritin is redox active, taking up six electrons per molecule of ferritin (Watt and Frankel, 1990; Watt et al., 1992). This has led us to question the importance of this characteristic to the overall function of ferritin. By understanding how the redox state of the protein affects the protein function in vitro we can better understand how this function, iron binding, occurs in vivo.
I have performed experiments designed to help us understand the redox function of ferritin and its importance to ferritin function. By investigating the metal ion binding to ferritin in both its reduced and oxidized states we can see the effect this redox characteristic has on the protein function.
To determine the effect of the redox state of the protein on its iron binding function I performed iron binding and zinc binding studies. I determined iron and zinc binding to reduced and oxidized apoferritin from horse spleen (HoSF). These studies were carried out as described in Pead, et. al. (1995) and Jacobs, et. al. (1989).
In agreement with previous findings (Pead, et. al., 1995), I found that reduced apoferritin binds 8±0.5 iron atoms (Fe ) per ferritin protein 2+ molecule (HoSF). The oxidized ferritin, however bound many times more than the reduced, 30±10 Fe2+/HoSF. The zinc binding to reduced apoferritin resulted in 31±5 Zn2+/HoSF, the same as the reported value for zinc binding to oxidized apoferritin (Pead, et. al., 1995). Table 1 shows the comparison of the binding results.
These results demonstrate two important characteristics of ferritin. First, that the redox state of the protein affects the iron binding of the protein. This is very clear as three times more iron bound to the oxidized ferritin than to the reduced. Second, the redox state only affects iron binding, or is iron specific. This is demonstrated by the absence of any change in the zinc binding from the reduced to the oxidized ferritin. These results indicate that the redox state of the protein does play a role in the iron uptake function of ferritin.
A model for ferritin function is also suggested by the results. Channels to the interior of the protein shell are found in the ferritin tertiary structure. Eight of these channels are located at the three-fold subunit axis and are hydrophilic. The eight iron atoms bound to the reduced ferritin most likely are bound within these channels, one per channel. While bound within a channel, the iron must prevent further iron entry to that channel and the protein interior. Yet, when the iron is bound to the oxidized ferritin electrons can be transferred from the iron to the protein, oxidizing the iron and allowing the iron to enter the core. In this manner more iron (30 Fe/HoSF) can bind to the oxidized ferritin. This model may explain a small effect of the redox state of the protein on its iron binding function.
References
- Harrison, P. M., Andrews, S. C., Artymiuk, P. J., Ford, G. C., Guest, J. R., Hirzmann, J., Lawson, D. M., Livingstone, J. C., Smith, J. M. A.,
Treffry, A., & Yewdall, S. J. (1991) Adv. Inorg. Chem. 36, 449-486. - Jacobs, D., Watt, G. D., Frankel, R. B., & Papaefthymiou, G. C. (1989) Biochemistry 28, 9216-9221.
- Pead, S., Durrant, E., Webb, B., Larsen, C., Heaton, D., Johnson, J., & Watt, G. D. (1995) J. Inorg. Biochem. 59, 15-27.
- Theil, E. C. (1990) Adv. Enzymol. 63, 421-449.
- Watt, G. D., & Frankel, R. B. In Iron Biominerals; Frankel, R. B., & Blakemore, R. P. Eds.; Plenum Press: New York, 1990; p.307-313.
- Watt, R. K., Frankel, R. B., & Watt, 6 G. D. (1992) Biochemistry 31, 9673-9679.