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

Abstract

Horse spleen ferritin will bind up to 16 protoporphyrin IX haem groups per 24 subunits in vitro [Kadir & Moore (1990) FEBS Lett. 276, 81-84] at a site that causes the haem to be low spin for both ferric and ferrous states. E.p.r. spectra at 10 K of the oxidized form of the resulting haemoferritin gives g values of 2.93, 2.26 and 1.55, characteristic of low-spin haem. The near-i.r. magnetic circular dichroism spectrum shows a porphyrin-to-ferric charge-transfer band at 1590 nm. The spectroscopic parameters indicate that the haem group is probably bound by two histidine ligands. Molecular modelling studies reveal one type of potential haem-binding site in horse L-chain ferritin with bis-histidine co-ordination. This is an intersubunit site which lies in a pocket within the ferritin protein shell in the region of the 3-fold channel. The ligands are His-114 and His-124 in horse L-chain. A second possible set of sites in human H-chain ferritin involves His-60 residues in the pockets between pairs of subunits. These are considered less likely sites of haem occupancy. There are three of the intersubunit sites in horse L-chain ferritin at each of the eight 3-fold channels. We propose that conformational crowding between haem-binding sites at a given channel prevents more than two haems per channel being bound.

Optical and EPR spectroscopic studies of demetallation of hemin by L-chain apoferritins

“…However, in vitro studies showed that it was also possible to incorporate hemin (Fe(III)-PPIX) into horse spleen apoferritin [21]. From electron paramagnetic resonance (EPR), near-infrared magnetic circular dichroism (NIR-MCD) and molecular modelling studies, it was suggested that the heme-binding site was situated around the 3-fold symmetry axes and that the ligands of the heme iron were His 114 and His 124 [22]. However, crystallographic studies of horse spleen apoferritin cocrystallised with Sn(II)-protoporphyrin IX (PPIX) or with Fe(III)-PPIX (hemin), showed that the metal-free PPIX was bound in a hydrophobic pocket along the 2-fold symmetry axes [1,23].…”

A crystallographic study of haem binding to ferritin

“…Previous spectroscopic and molecular modelling results (Moore et al, 1992) suggested the haem group is probably bound by two histidine ligands, in the region of the threefold axis. Nothing in the structure of the complex reported here suggests that there are two kinds of haem-binding sites in a ferritin molecule.…”

“…This is the first report of a crystallographic study of the haem-binding site in ferritin, even if, during the writing of this paper, the low-resolution structure of bacterioferritin has been solved providing evidence of a nearly similar location of the haem molecule in this structurally related protein (Yariv, 1993). Besides these results, the structure of mammalian ferritin offers no indication of a haembinding site (Moore et al, 1992).…”

“…Not only the location of both the haem-binding site and the ferroxidase centre, but also the stoichiometry of haem binding to HSF and to bacterioferritin have been discussed in many recent publications (Smith et al, 1989;Kadir & Moore, 1990b;Grossman, Hinton, Minak-Bernero, Slaughter & Stiefel, 1992;Moore et al, 1992;Lawson et al, 1989Lawson et al, , 1991. Comparable binding properties were found in apo-myoglobin suggesting that the haem-binding site is rather non-specific (Gibson & Antonini, 1966).…”

Structural investigation of the complexation properties between horse spleen apoferritin and metalloporphyrins

“…'^ A first location of the possible binding sites, in ferritin, has been proposed by molecular modeling and spectroscopic investigations. 16 To describe these binding properties further, preliminary results of a crystallographic study17 of HSF co-crystallized with Snprotoporphyrin IX (PDB entry code: 1HRS) allowed us to assign clearly the location of the binding site among the two previously16 predicted sets of sites.…”