PROTEINS: Structure, Function, and Genetics 24:314-321 (1996) Structural Investigation of the Complexation Properties Between Horse Spleen Apoferritin and Metalloporphyrins Marie-Anges Michaux, Alain Dautant, Bernard Gallois, Thierry Granier, Beatrice Langlois d'Estaintot, and Gilles Precigoux Laboratoire de Cristallographie et de Physique Cristalline, ERS 133 CNRS, Universite de Bordeaux I, 33405 Talence Cedex, France ABSTRACT Crystallographic studies of L-chain horse spleen apoferritin (HSF)co-crys- tallized with Pt-hematoporphyrin IX and Sn- protoporphyrin IX have brought significant new insights into structure-function relation- ships in ferritins. Interactions of HSF with por- phyrins are discussed. Structural results show that the nestling properties into HSF are depen- dent on the porphyrin moiety. (Only protopor- phyrin IX significantly interacts with the pro- tein, whereas hematoporphyrin IX does not.) These studies additionally point out the L-chain HSF ability to demetalate metalloporphyrins, a result which is of importance in looking at the iron storage properties of ferritins. In both compound investigated (whether the porphyrin reaches the binding site or not), the complex- ation appears to be concomitant with the ex- traction of the metal from the porphyrin. To analyze further the previous results, a three-dimensional alignment of ferritin se- quences based on available crystallographic coordinates, including the present structures, is given. It confirms a high degree of homology between these members of the ferritin family and thus allows us to emphasize observed structural differences: 1) unlike L-chain HSF, H-chain human ferritin presents no preformed binding site; and 2) despite the absence of axial ligands, and due to the demetalation, L-chain HSF is able to host protoporphyrin at a similar location to that naturally found in bacteriofer- ritin. o 19% Wiley-Liss, Inc. Key words: X-ray protein crystallography, structure determination, anoma- lous scattering, protein-porphyrin interaction, metalloporphyrin de- metalation, multiple alignment, structural comparison, ferritin and bacterioferritin was first isolated from mammalian spleen and liver by Laufberger' in 1937. Natural ferritin was found to comprise two (H and L) subunits, sharing 55% amino acid sequence identity. More than 35 se- quences of ferritins and bacterioferritins are fully or partially Structure similarities of both eu- karyotic ferritins (horse ~ p l e e n , ~ rat liver,5 recombi- nant rat L,6 b~llfrog,~ and human H ferritin5) and prokaryotic ferritin (E. coli bacterioferritin') were extensively demonstrated. Each ferritin subunit is a bundle of four (A, B, C, D) a-helices with a short fifth (E) a-helix at the C terminus and a loop connecting the antiparallel helix pairs A-B and C-D. The three- dimensional arrangement obeys a 432 symmetry. Each molecule (24 identical subunits) forms a hol- low shell of nearly 80 A in diameter, conveniently described as a truncated rhombic dodecahedron into which iron is deposited as mineral ferrihydrite.' Whereas bacterioferritin binds in vivo, eukaryotic ferritins are known as a non-heme iron pr~tein.'~,'~ However, in in vitro experiments, using spectroscopic techniques, Kadir and Moore14showed that horse spleen ferritin (HSF) is able to interact with heme. This result is of importance: it reinforces the similarity between the two proteins and also suggests that heme may be an integral part of eu- karyotic ferritin and not only of bacterioferritin, since heme is known to act as an inducer in the expression of ferritin in vertebrate^.'^ A first loca- tion of the possible binding sites, in ferritin, has been proposed by molecular modeling and spectro- scopic investigations.16 To describe these binding properties further, preliminary results of a crystal- lographic study17 of HSF co-crystallized with Sn- protoporphyrin IX (PDB entry code: 1HRS) allowed us to assign clearly the location of the binding site among the two previously16 predicted sets of sites. We present here a more complete description of the complexation properties, enhancing the fact that the metal ions are extracted from the metallopor- INTRODUCTION The ability of ferritin to take up and release iron atoms reversibly has been widely studied since it 0 1996 WILEY-LISS, INC Received May 19, 1995; revision accepted October 18, 1995. Address reprint requests to Bernard Gallois, Laboratoire de Cristallographie et de Physique Cristalline, Universite de Bor- deaux I, 33405 Talence Cedex, France.