Protein Association and Dissociation Regulated by Ferric Ion

Abstract

Iron stored in phytoferritin plays an important role in the germination and early growth of seedlings. The protein is located in the amyloplast where it stores large amounts of iron as a hydrated ferric oxide mineral core within its shell-like structure. The present work was undertaken to study alternate mechanisms of core formation in pea seed ferritin (PSF). The data reveal a new mechanism for mineral core formation in PSF involving the binding and oxidation of iron at the extension peptide (EP) located on the outer surface of the protein shell. This binding induces aggregation of the protein into large assemblies of ∼400 monomers. The bound iron is gradually translocated to the mineral core during which time the protein dissociates back into its monomeric state. Either the oxidative addition of Fe2+ to the apoprotein to form Fe3+ or the direct addition of Fe3+ to apoPSF causes protein aggregation once the binding capacity of the 24 ferroxidase centers (48 Fe3+/shell) is exceeded. When the EP is enzymatically deleted from PSF, aggregation is not observed, and the rate of iron oxidation is significantly reduced, demonstrating that the EP is a critical structural component for iron binding, oxidation, and protein aggregation. These data point to a functional role for the extension peptide as an iron binding and ferroxidase center that contributes to mineralization of the iron core. As the iron core grows larger, the new pathway becomes less important, and Fe2+ oxidation and deposition occurs directly on the surface of the iron core. Iron stored in phytoferritin plays an important role in the germination and early growth of seedlings. The protein is located in the amyloplast where it stores large amounts of iron as a hydrated ferric oxide mineral core within its shell-like structure. The present work was undertaken to study alternate mechanisms of core formation in pea seed ferritin (PSF). The data reveal a new mechanism for mineral core formation in PSF involving the binding and oxidation of iron at the extension peptide (EP) located on the outer surface of the protein shell. This binding induces aggregation of the protein into large assemblies of ∼400 monomers. The bound iron is gradually translocated to the mineral core during which time the protein dissociates back into its monomeric state. Either the oxidative addition of Fe2+ to the apoprotein to form Fe3+ or the direct addition of Fe3+ to apoPSF causes protein aggregation once the binding capacity of the 24 ferroxidase centers (48 Fe3+/shell) is exceeded. When the EP is enzymatically deleted from PSF, aggregation is not observed, and the rate of iron oxidation is significantly reduced, demonstrating that the EP is a critical structural component for iron binding, oxidation, and protein aggregation. These data point to a functional role for the extension peptide as an iron binding and ferroxidase center that contributes to mineralization of the iron core. As the iron core grows larger, the new pathway becomes less important, and Fe2+ oxidation and deposition occurs directly on the surface of the iron core. The chemistry of iron and oxygen in a number of non-heme di-iron proteins has been a subject of intense interest because of their varied roles in oxygen activation and catalysis, substrate hydrolysis, oxygen transport, redox reactions, H2O2, and iron detoxification and iron storage. Di-iron centers have similar structural motifs consisting of a combination of carboxylate and histidine ligands that either bind or bridge the two metal ions of the di-nuclear active site; di-iron proteins containing these centers include methane monooxygenase, ribonucleotide reductase, rubrerythrin, stearoyl desaturase, purple acid phosphatase, hemerythrin, the Dps proteins, and ferritins (1Harrison P.M. Arosio P. Biochim. Biophys. Acta. 1996; 1275: 161-203Crossref PubMed Scopus (2278) Google Scholar, 2Chasteen N.D. Harrison P.M. J. Struct. Biol. 1999; 126: 182-194Crossref PubMed Scopus (682) Google Scholar, 3Zhao G. Ceci P. Ilari A. Giangiacomo L. Laue T.M. Chiancone E. Chasteen N.D. J. Biol. Chem. 2002; 277: 27689-27696Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 4Solomon E.I. Brunold T.C. Davis M.I. Kemsley J.N. Lee S.K. Lehnert N. Neese F. Skulan A.J. Yang Y.S. Zhou J. Chem. Rev. 2000; 100: 235-350Crossref PubMed Scopus (1529) Google Scholar, 5Kolberg M. Strand K.R. Graff P. Andersson K.K. Biochim. Biophys. Acta. 2004; 1699: 1-34Crossref PubMed Scopus (254) Google Scholar, 6Kopp D.A. Lippard S.J. Curr. Opin. Chem. Biol. 2002; 6: 568-576Crossref PubMed Scopus (109) Google Scholar). Despite their similar di-nuclear centers, each of these proteins fulfills a distinct biological role that seems to be mediated by the nature of the first and second coordination sphere of the di-iron center. Ferritins are a class of intracellular iron storage and detoxification proteins that facilitate the oxidation of iron by molecular oxygen or hydrogen peroxide to form a hydrous ferric oxide mineral core within their interiors (1Harrison P.M. Arosio P. Biochim. Biophys. Acta. 1996; 1275: 161-203Crossref PubMed Scopus (2278) Google Scholar, 2Chasteen N.D. Harrison P.M. J. Struct. Biol. 1999; 126: 182-194Crossref PubMed Scopus (682) Google Scholar, 3Zhao G. Ceci P. Ilari A. Giangiacomo L. Laue T.M. Chiancone E. Chasteen N.D. J. Biol. Chem. 2002; 277: 27689-27696Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). Rapid Fe2+ oxidation occurs at the di-iron ferroxidase center located on the H-subunit of the mammalian protein. Unlike the H-chain, the more acidic L-subunit lacks the ferroxidase center but contains a putative nucleation site responsible for slower iron oxidation and mineralization (7Crichton R.R. Herbas A. Chavez-Alba O. Roland F. J. Biol. Inorg. Chem. 1996; 1: 567-574Crossref Scopus (47) Google Scholar). The shapes of both the H- and L-subunit are nearly cylindrical and composed of a four-α-helix bundle containing two antiparallel helix pairs (A, B and C, D) connected by a long non-helical stretch, the BC-loop, between B and C helices. A fifth short helix (E helix) lies at one end of the bundle at 60° to its axis (1Harrison P.M. Arosio P. Biochim. Biophys. Acta. 1996; 1275: 161-203Crossref PubMed Scopus (2278) Google Scholar, 2Chasteen N.D. Harrison P.M. J. Struct. Biol. 1999; 126: 182-194Crossref PubMed Scopus (682) Google Scholar). From an evolutionary point of view, plant and animal ferritins arose from a common ancestor, but plant ferritins exhibit various specific features as compared with animal ferritins. Plant ferritins are observed in plastids (chloroplasts in leaves, amyloplasts in tubers and seeds, etc.) where iron is incorporated into the ferritin shell to form the mineral core, whereas animal ferritins are largely found in the cytoplasm of cell (1Harrison P.M. Arosio P. Biochim. Biophys. Acta. 1996; 1275: 161-203Crossref PubMed Scopus (2278) Google Scholar, 8Waldo G.S. Wright E. Whang Z.H. Briat J.F. Theil E.C. Sayers D.E. Plant Physiol. 1995; 109: 797-802Crossref PubMed Scopus (88) Google Scholar). Ferritins from dried soybean and pea seed consist of two subunits of 26.5 and 28.0 kDa, which are designated H-1 and H-2, respectively, share ∼80% amino acid sequence identity (9Masuda T. Goto F. Yoshihara T. J. Biol. Chem. 2001; 276: 19575-19579Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 10Laulhere J.P. Laboure A.M. Briat J.F. J. Biol. Chem. 1989; 264: 3629-3635Abstract Full Text PDF PubMed Google Scholar), and contain ferroxidase centers. The two subunits are synthesized from a 32-kDa precursor with a unique two-domain N-terminal sequence containing a transit peptide (TP) 2The abbreviations used are: TPtransient peptideEPextension peptideMops3-(N-morpholino)propanesulfonic acidDLSdynamic light scatteringPSFpea seed ferritinapoPSFapo pea seed ferritin. and an extension peptide (EP). The TP is responsible for precursor targeting to plastids (11Ragland M. Briat J.F. Gagnon J. Laulhere J.P. Massenet O. Theil E.C. J. Biol. Chem. 1990; 265: 18339-18344Abstract Full Text PDF PubMed Google Scholar). Upon transport to plastids, the TP is cleaved from the subunit precursor, resulting in the formation of the mature subunit which assembles into a 24-mer apoferritin within the plastids (11Ragland M. Briat J.F. Gagnon J. Laulhere J.P. Massenet O. Theil E.C. J. Biol. Chem. 1990; 265: 18339-18344Abstract Full Text PDF PubMed Google Scholar, 12Lescure A.M. Proudhon D. Pesey H. Ragland M. Theil E.C. Briat J.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8222-8226Crossref PubMed Scopus (153) Google Scholar). The EP domain is kept at the N-terminal extremity of the mature plant ferritins, but its function remains unknown. It is absent in animal ferritins. For soybean ferritin, further processing of the 26.5-kDa subunit occurs through excising a short C-terminal amino acid sequence (16 amino acid residues) that corresponds to the E helix of the mammalian ferritin subunit (9Masuda T. Goto F. Yoshihara T. J. Biol. Chem. 2001; 276: 19575-19579Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Compared with their own precursors, H-1 is devoid of both the N-terminal TP domain and the C-terminal E helix, whereas H-2 lacks the N-terminal TP domain only (13Masuda T. Goto F. Yoshihara T. Ezure T. Suzuki T. Kobayashi S. Shikata M. Utsumi S. Protein Expr. Purif. 2007; 56: 237-246Crossref PubMed Scopus (36) Google Scholar). However, the amino acids constituting the ferroxidase center are strictly conserved in all the plant ferritins except for pea seed ferritin where His-62 replaces Glu-62 (14van Wuytswinkel O. Savino G. Briat J.F. Biochem. J. 1995; 305: 253-261Crossref PubMed Scopus (65) Google Scholar, 15Lobreaux S. Yewdall S.J. Briat J.F. Harrison P.M. Biochem. J. 1992; 288: 931-939Crossref PubMed Scopus (83) Google Scholar). transient peptide extension peptide 3-(N-morpholino)propanesulfonic acid dynamic light scattering pea seed ferritin apo pea seed ferritin. Iron oxidation/mineralization in human ferritin occurs by at least three reaction pathways (16Zhao G. Bou-Abdallah F. Arosio P. Levi S. Janus-Chandler C. Chasteen N.D. Biochemistry. 2003; 42: 3142-3150Crossref PubMed Scopus (140) Google Scholar, 17Yang X. Chen-Barrett Y. Arosio P. Chasteen N.D. Biochemistry. 1998; 37: 9743-9750Crossref PubMed Scopus Google Scholar). Fe2+ binding at the ferroxidase the oxidation of Fe2+ is the of and a mineral core of Fe3+ is for as A. Harrison P.M. Biochemistry. PubMed Scopus Google Scholar, Chasteen N.D. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). of the in with Fe2+ in the Fe2+ detoxification reaction to (16Zhao G. Bou-Abdallah F. Arosio P. Levi S. Janus-Chandler C. Chasteen N.D. Biochemistry. 2003; 42: 3142-3150Crossref PubMed Scopus (140) Google Scholar, G. Arosio P. Chasteen N.D. Biochemistry. PubMed Scopus Google Scholar). a mineral core of has Fe2+ becomes and iron oxidation and occurs on the surface of the mineral through an where is to (1Harrison P.M. Arosio P. Biochim. Biophys. Acta. 1996; 1275: 161-203Crossref PubMed Scopus (2278) Google Scholar, R.R. Herbas A. Chavez-Alba O. Roland F. J. Biol. Inorg. Chem. 1996; 1: 567-574Crossref Scopus (47) Google Scholar, 17Yang X. Chen-Barrett Y. Arosio P. Chasteen N.D. Biochemistry. 1998; 37: 9743-9750Crossref PubMed Scopus Google Scholar). on the and on of by and the mechanism of mineral core formation in mammalian ferritins has been A. Harrison P.M. Biochemistry. PubMed Scopus Google Scholar, J. C. D.E. Theil E.C. 2000; PubMed Scopus Google Scholar, F. G. Arosio P. Chasteen N.D. J. Chem. PubMed Scopus Google Scholar, C. P. D.E. Theil E.C. Biochemistry. 1998; 37: PubMed Scopus Google Scholar). two Fe2+ are by at the ferroxidase center to form a transient which to form a to the The from the ferroxidase center to the to form an core, which to the formation of the mineral core However, mechanism of oxidative deposition of iron is only to ferritins as ferritin and human ferritin, which at their ferroxidase centers. the ferroxidase centers of human ferritin F. P. Levi S. Arosio P. Chasteen N.D. J. Biol. PubMed Scopus Google Scholar), X. A.J. Chasteen N.D. Biochemistry. 2000; PubMed Scopus Google Scholar), E. ferritin A A. Harrison P.M. 1998; PubMed Scopus Google Scholar), and pea seed ferritin C. X. G. PubMed Scopus Google and Fe3+ at their centers with from the center to the to form the core. These the as to alternate pathways for core formation in these proteins, in in the of the present study have for alternate mechanisms of iron deposition in PSF and that aggregation occurs during the oxidative deposition of iron in PSF more are to the apoprotein an the 24 ferroxidase center binding capacity for aggregation was by the addition of Fe3+ directly to The data that binding occurs on the outer surface of the ferritin shell at involving the extension Upon the iron dissociates to its whereas the iron to the mineral core. This an alternate pathway for iron deposition in seed ferritin was as C. X. G. PubMed Scopus Google Scholar). was in A. Harrison P.M. 1998; PubMed Scopus Google Scholar, Harrison P.M. D. A. Biochim. Biophys. Acta. 1991; PubMed Scopus Google by the in the and apoferritin to the as the pea seed ferritin with of iron core was as J. Chasteen N.D. G. J. Chem. 2002; PubMed Scopus Google Scholar). The EP of PSF with sequence was from was more as by and pea seed ferritin was to of and at for by at a of to the the pea seed ferritin subunit with the EP the was with and to its an was to the of the protein as in The N-terminal amino acid of the two new subunits with the are in that EP been from The molecular of the pea seed ferritin was by a at for at and as with was with as by PubMed Scopus Google Scholar). Protein in to which was of containing and the was for the was by at for The amino acid sequence of the was on a protein protein was to a and with The two subunits from the and the sequence of the amino acids at the was for each light scattering with a in with a protein aggregation by of a acidic or and with of iron core at in the containing an are the two The time was to be the and acid reaction H. M. J. Biochem. PubMed Scopus Google Scholar). and at and the in scattering light at a to the was E. H. J. Biol. PubMed Scopus Google Scholar). The data with The of PSF aggregation from the of a to the and is the time in and is the in of and on ferritin aggregation by ferric observed with the at with both and at E. H. J. Biol. PubMed Scopus Google Scholar). The in scattering was Fe2+ or with of apoferritin in When the scattering of of or was to of and The dynamic at a dynamic The the of the in the cell from the function of light The of the scattering is from the where is the the in and is the of the The protein was in containing at Protein through with or each was each for the The was from the the from which an molecular was to a from The was used to the of protein from the in or the addition of ferric all to for to that the the G. Ceci P. Ilari A. Giangiacomo L. Laue T.M. Chiancone E. Chasteen N.D. J. Biol. Chem. 2002; 277: 27689-27696Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, F. P. Levi S. Arosio P. Chasteen N.D. J. Biol. PubMed Scopus Google Scholar). The of the EP was in and at The by of Fe3+ to of the each Fe3+ Iron oxidation by the at the on a at C. X. G. PubMed Scopus Google Scholar). of iron and ferritin and time with the The was in and each with a containing apoferritin in of alternate mechanisms of iron deposition in PSF, for protein during Fe2+ study in dynamic and light scattering The in which Fe2+ was with apoPSF The light scattering within the time of the was with the apoprotein found in of the iron the light scattering to protein aggregation apoferritin was with a of Fe2+ to from to the rate and of the a function of the the 24 di-nuclear ferroxidase centers of PSF have a binding capacity of Fe3+ and are located in the of the protein C. X. G. PubMed Scopus Google Scholar), these that is by binding of the iron on the surface of protein shell of the are and contain an to the oxidation of Fe2+ at of to apoPSF and pea seed ferritin containing from to The data that the of light the of aggregation as the core in an with iron binding and oxidation on the surface of the mineral in on the of the protein shell. light scattering to Fe2+ or Fe3+ is responsible for the observed protein aggregation. scattering Fe2+ is to apoPSF to Fe3+ whereas the addition of Fe2+ causes A in was observed Fe3+ was directly the The for addition of Fe2+ to apoPSF is and for the reaction to to the to Fe2+ When Fe3+ is directly to the the is and the reaction is within Fe3+ not with a in scattering was observed the addition of amounts of Fe3+ to apoPSF from to The in a with a of are more and by a that to be first in iron The for the was to the reaction in A a monomeric in which Fe3+ is bound to the outer surface of the ferritin shell that to form C, and of The light scattering as a function of time was to the where the are for the light scattering of C, and at the and and are their as a function of time as F. G. Arosio P. Chasteen N.D. J. Chem. PubMed Scopus Google Scholar). and and and The scattering the as for of similar with protein from three protein that the observed are not a significantly to the data and large with the A B C with the reaction B a between the and of these The the at of and The of and observed with these as for the Protein by Fe3+ was with light scattering its at of Fe3+ to apoPSF and remains for more not to for at at with of and are in the light of the apoPSF The that the at is in of the whereas the second a of composed of This that at all of apoPSF in a state. the is large of to apoPSF and The with of the and corresponds to a large of ∼400 of PSF in protein with that Fe3+ the formation of large protein a in with the of and light scattering from of of apoPSF and apoPSF the as used in each of the in the of to the of is of number of monomeric PSF that the A molecular of is each of the in the of to the of is number of monomeric PSF that the A molecular of is in a new the that iron to the apoprotein in of the to the 24 ferroxidase centers C. X. G. PubMed Scopus Google on the outer surface of the protein where it protein aggregation. apoPSF and the light scattering a at was to the at a iron of The causes a in the light scattering to within a was used of and in the light scattering was observed that the by is not from the of with pea seed ferritin, in light or in from iron in the mineral core was observed the addition of that iron located in the of the protein is not by the iron responsible for aggregation is bound on the surface of the protein where and further the of protein in Fe2+ oxidation, a of rate of Fe2+ oxidation by oxygen as a function of protein was at a Fe2+ of or at a of both the of iron oxidation a on protein of a a between iron oxidation and protein The for PSF that the EP is located on the outer surface of protein S. Yewdall S.J. Briat J.F. Harrison P.M. Biochem. J. 1992; 288: 931-939Crossref PubMed Scopus (83) Google Scholar), the of the peptide contains the binding for Fe2+ and apoPSF with the EP deleted was with Fe2+ or in the to PSF, in the light scattering was observed with the protein a that the extension peptide is an structural component for iron binding and protein aggregation. the the EP was and a binding study with Fe3+ The of the EP containing amounts of Fe3+ are in occurs at for the that of the observed is by the at The of that Fe3+ to the EP with a a the as the light scattering in Fe2+ oxidation in both apoPSF and apoPSF was by at as C. X. G. PubMed Scopus Google Scholar). is observed during the time of the in the light scattering of The in is to Fe2+ oxidation at the ferroxidase site C. X. G. PubMed Scopus Google Scholar, G. M. Chasteen N.D. J. Biol. PubMed Scopus Google Scholar, H. C. A. A. J. Biol. Inorg. Chem. 2004; PubMed Scopus Google as is nearly with both PSF and PSF because both have ferroxidase centers. However, iron oxidation in the second by the protein is that of EP deleted PSF, that the EP Fe2+ oxidation in As the iron into ferritin from to the in iron oxidation rate between the two proteins becomes not These are with the in to Fe2+ oxidation by the EP of the protein structure. ferritin during the oxidative deposition of iron in the protein is a was in which shell with The in was by a a of to its with in light was an in at because of growth in the mineral core the of protein by iron The and of are in and was observed with by Fe3+ light by addition of Fe2+ to apoPSF at apoPSF in The present work that PSF has not the aggregation of PSF is by the substrate for ferritin. The of is only observed the addition of more to a of the of the ferroxidase center of PSF C. X. G. PubMed Scopus Google Scholar), the Fe2+ to bind at where it is the in the light scattering This oxidation for and is the for Fe2+ these not PSF contains at least two of centers for Fe2+ one is located on the outer and the is located at the ferroxidase centers. with view, the to PSF aggregation by ferric are and the by Fe2+ and The observed by Fe2+ Fe3+ that PSF aggregation is only by the As aggregation not with PSF shell are to apoPSF protein aggregation is by Fe3+ only more shell are to apoPSF further that it is Fe3+ bound at that The addition of in at a rate compared with because of of the Fe3+ to in the protein by Fe3+ was by a aggregation by in which B and C of are to the D. for the from that with for the of the second in apoPSF to of the by a in the first at to A. The at contains the B and C, whereas the corresponds to the with and is composed of an of ∼400 of the EP from PSF protein aggregation in the of Fe3+ demonstrating that the EP is an structural component of the iron binding that synthesized EP Fe3+ with a PSF a significantly rate of iron oxidation in the second as compared with the ferritin that the EP domain not only in the binding of iron but its oxidation as a second center of iron oxidation in addition to the ferroxidase center. The of the EP of the subunit is by and for nearly of the 24 EP and is back on the outer surface of the subunit S. Yewdall S.J. Briat J.F. Harrison P.M. Biochem. J. 1992; 288: 931-939Crossref PubMed Scopus (83) Google Scholar). It contains two and and one in to one but S. Yewdall S.J. Briat J.F. Harrison P.M. Biochem. J. 1992; 288: 931-939Crossref PubMed Scopus (83) Google Scholar). These three acidic ligands for Fe3+ binding in which ferritin are through coordination to iron to form the because of its and is Fe2+ for binding at these and aggregation. PSF is and the more by the binding of Fe3+ to Fe2+ plays role in protein Protein aggregation by the addition Fe2+ is a function of the of the and is was observed with protein aggregation from the direct addition of Fe3+ not of the occurs with an in from growth of the mineral core on these a new pathway to iron oxidative deposition in ferritin through by Fe3+ is as in is the binding reaction of Fe2+ with the EP domain located on the surface of PSF at Fe2+ oxidation by the of the EP the second to A. The resulting bound Fe3+ protein through B and C, PSF as by which corresponds to Fe3+ is from the outer surface of protein to the of the into is the It is that pathway is with both and and only the of iron is in of that to the 24 ferroxidase centers. However, pathway is distinct from the mineralization reaction to where Fe2+ directly occurs on the surface of the core. As the of iron core the mineral surface reaction becomes important as X. Chen-Barrett Y. Arosio P. Chasteen N.D. Biochemistry. 1998; 37: 9743-9750Crossref PubMed Scopus Google Scholar, Chasteen N.D. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar, A. Harrison P.M. 1998; PubMed Scopus Google Scholar), and as is less iron aggregation the protein a core of aggregation was observed The present are in with of Harrison and Harrison P.M. D. A. Proc. Biol. Sci. 1991; PubMed Scopus Google and ferritin Fe3+ from the to the and and large mineral at the of of iron through as found in the present The aggregation observed for PSF not from or as for ferritins during the oxidative deposition of iron in these proteins S. Biophys. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Biochemistry. PubMed Scopus Google Scholar, Biol. 2001; PubMed Scopus Google Scholar). and observed in was for protein in or of apoPSF with compared with apoPSF not the present study that the addition of Fe2+ or Fe3+ to PSF with its ferroxidase centers with iron (48 in protein by the binding of Fe3+ to the outer surface of the protein at the extension This iron is in the mineral core by a that a new mechanism for the oxidative deposition of iron in ferritin. with