Remarkable ability of horse spleen apoferritin to demetallate hemin and to metallate protoporphyrin IX as a function of pH.
In previous studies it has been shown that reaction of crystalline horse spleen apoferritin with hemin leads to a protoporphyrin IX-apoferritin complex [Précigoux et al. (1994) Acta Crystallogr. D50, 739-743]. We show here the following. (i) Hemin binds to two classes of sites in horse spleen apoferritin at pH 8, each with a binding stoichiometry of 0.5 hemin/subunit; protoporphyrin IX also binds to horse spleen apoferritin with an apparent binding stoichiometry of 1 molecule of protoporphyrin IX/subunit. (ii) When Fe(III)-protoporphyrin IX binds to apoferritin, there is a pH-dependent loss of the metal ion, extremely slow at alkaline pH values (half-time of weeks) and much more rapid at acidic pH values (half-time of seconds below pH 5.0); maximum rates of demetallation are found at pH 4.0, and at lower pH values they decrease. (iii) Chemical modification of 11 carboxyl groups/subunit in horse spleen apoferritin does not affect hemin binding at alkaline pH values; however, it prevents hemin demetallation at acidic pH values. (iv) Hemin that has been demetallated at acidic pH values can be remetallated by increasing the pH; the rate of remetallation is greater at more alkaline pH values. (v) When around 20 atoms of iron/molecule are incorporated into horse spleen apoferritin and protoporphyrin IX is then bound, iron can subsequently be transferred to the porphyrin at pH 8.0. A mechanism is proposed to explain demetallation of heme, involving attack on the tetrapyrrole nitrogens of the protoporphyrin IX-Fe by protons derived from protein carboxylic acid groups and subsequent complexation of the iron by the corresponding carboxylates and binding of protoporphyrin IX to a preformed pocket in the inner surface of the apoferritin protein shell. The cluster of carboxylates involved is situated at the entrance to the pocket in which the protoporphyrin IX molecule is bound and has been previously identified as the site of iron incorporation into L-chain apoferritins. This appears to be the first example of iron removal and incorporation into porphyrins under relatively mild physiological conditions.
Crichton
R R
RR
Unité de Biochimie, Université Catholique de Louvain, place Louis Pasteur 1, B-1348 Louvain-la-Neuve, Belgium.
Soruco
J A
JA
Roland
F
F
Michaux
M A
MA
Gallois
B
B
Précigoux
G
G
Mahy
J P
JP
Mansuy
D
D
D000818:Animals; D001052:Apoferritins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D006427:Hemin / Q000737:chemistry*; D006736:Horses; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000737:chemistry; D011524:Protoporphyrins / Q000737:chemistry* / Q000378:metabolism; D013154:Spleen / Q000737:chemistry*
D008670:Metals; D011524:Protoporphyrins; D006427:Hemin; D001052:Apoferritins; C028025:protoporphyrin IX