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).(ABSTRACT TRUNCATED AT 250 WORDS)
Zhang
J H
JH
Department of Chemistry, University of Georgia, Athens 30602.
Kurtz
D M
DM
Xia
Y M
YM
Debrunner
P G
PG
D000818:Animals; D001059:Apoproteins; D006422:Hemerythrin / Q000031:analogs & derivatives* / Q000648:ultrastructure*; D066298:In Vitro Techniques; D007448:Invertebrates; D007501:Iron; D013056:Spectrophotometry, Ultraviolet; D013057:Spectrum Analysis
D001059:Apoproteins; D006422:Hemerythrin; C008571:myohemerythrin; D007501:Iron