Haem binding to horse spleen ferritin and its effect on the rate of iron release

Abstract

Horse spleen ferritin is shown to bind haem to generate a haemoprotein, named herein haemoferritin. A total of 14-16 haem molecules are bound per 24 subunits of ferritin. The molecular mass of the non-haem-iron-free haemoferritin has been determined to be 420 + 40 kDa, indicating that haem binding does not lead to dissociation of the 24 subunits that comprise the ferritin molecule. The functional role of the bound haem has been investigated with respect to the release of iron from the non-haem iron core. The bound haem is shown to increase the rate of iron release in a reductive assay system. In the absence of haem the rate of iron release depends on the redox potential of the reductant, but in the presence of haem the rate is largely independent of the reductant and is faster than the rate for the haem-free ferritin. These data indicate that electron transfer across the protein coat of ferritin is the rate-limiting step of iron release in the absence of haem, but in the presence of haem electron transfer is not rate-limiting.

Ferritins: furnishing proteins with iron

“…This can be seen by considering the basic scheme of the commonly employed in vitro reductive iron release assays (Eqs. 1 , 2 ) [ 83 – 85 ]: …”

“…5 ), it appears that electron transfer through the ferritin protein is important in reductive release of Fe 2+ ions, at least in the small molecule studies using the assays of reactions ( 1 ) and ( 2 ). This is shown by the dependence of the overall rate of formation of Fe 2+ (L) x on the redox potential of the electron donor [ 84 , 85 ].…”

Model of crystallization in a confined space applied to iron uptake and release by ferritin

“…Iron release has been the focus of numerous studies that used either a strong reducing agent, such as dithionite, or other reducers, such as superoxide anions 0;- (Monteiro et al, 1989), reduced flavins (Sirivech et al, 1974;Imai et al, 1980), or viologens (Jacobs et al, 1989). It is not clear whether the reducing molecule penetrates the protein shell by some of the channels or if a long-range electronic transfer occurs through the shell (Kadir et al, 1991;Kadir et al, 1992).…”

Spectroscopic identification of the haem axial ligands of haemoferritin and location of possible haem-binding sites in ferritin by molecular modelling

“…A major difference between ferritin and BFR is that, as isolated, BFR contains haem and ferritin does not. However, recent optical spectroscopic studies of horse spleen ferritin have shown that in vitro it binds the protoporphyrin IX haem group to generate haemoferritin (Kadir & Moore, 1990;Kadir et al, 1992). Approx.…”

“…Horse spleen ferritin was obtained from the Sigma Chemical Co. (Poole, Dorset, U.K.) and the non-haem iron removed by dialysis against thioglycollic acid, at pH 4.25 and 4°C for 5 days followed by dialysis against 25 mM-phosphate buffer at pH 7 for 2 days. Haemoferritin was prepared from the resulting apoferritin by the procedure by Kadir et al (1992).…”