RECONSTITUTION OF THE DIIRON SITES IN HEMERYTHRIN AND MYOHEMERYTHRIN

The first reconstitutions of functional diiron sites in the nonheme O2-carrying proteins hemerythrin (Hr) and myohemerythrin (myoHr) have been achieved. Both proteins are reconstituted under anaerobic conditions, and the procedure consists of (i) denaturation of the native met form with 6 M guanidinium chloride in the presence of sodium dithionite and 2,2'-dipyridyl, (ii) separation of the apoprotein from the other reagents and products, (iii) addition of an iron(II) stock solution to the apoprotein in the presence of 2-mercaptoethanol, and (iv) several cycles of slow dilution and reconcentration by ultrafiltration to remove excess reagents. Iron analyses indicate that the apoproteins have been essentially completely freed of iron and that reconstituted Hr contains its full complement of iron, i.e., approximately 2 Fe/subunit. Ferrous rather than ferric iron appears to be necessary for recovery of the native structures for both myoHr and Hr. In the case of Hr, reconstitution was successful only when iron(II) was added to apoHr prior to removal of denaturant. ApoHr is essentially insoluble at pH 7 in the absence of denaturants but remains soluble when denaturant is removed in the presence of ferrous iron, which leads to recovery of the octameric structure containing all of its diiron sites. Iron(II) apparently stabilizes the native or a nearly native structure during reconstitution. OxymyoHr and oxyHr are the major initial products of reconstitution. The yield of oxymyoHr from apomyoHr was approximately 87%. In contrast to reconstituted oxymyoHr, where essentially all of the iron appears to be functional, approximately 30% of the diiron sites in the reconstituted oxyHr are unable to bind O2 at ambient p(O2). Mossbauer spectroscopy of Hr that was reconstituted with iron enriched in Fe-57 indicates that the nonfunctional iron is in an environment very similar to but not identical with that in native deoxyHr. The yield of reconstituted oxyHr from native metHr was approximately 72%, and this value includes both functional and nonfunctional iron. Oxidation of the reconstituted oxyHr with ferricyanide converts the protein to a form that is spectroscopically indistinguishable from native metHr, and the yield of this reconstituted metHr starting from native metHr was approximately 50%. The reconstitution procedures described here lay the groundwork for detailed examinations of the refolding process and for preparation of metal-substituted Hrs.