Conversion of non-functional to functional iron following reconstitution of hemerythrin.
A recent report from this laboratory (Zhang, J.-H., Kurtz, D.M., Jr., Xia, Y.-M. and Debrunner, P.G. (1991) Biochemistry 30, 583-589) described a procedure for reconstitution of a functional di-iron site in the octameric, non-heme iron O2-carrying protein, hemerythrin by addition of ferrous salts to apoprotein, followed by slow dilution of the denaturant. Although the resulting protein contained its full complement of iron, i.e., 2 Fe per subunit, about 30% of the iron was found to remain ferrous under ambient O2, i.e., this iron was incapable of forming an O2 adduct. In this report a method is described for obtaining essentially fully functional hemerythrin by passage of the freshly reconstituted protein through an [oxy/30% non-functional----met----deoxy----oxy redox cycle. UV/vis absorption and 57Fe Mössbauer spectroscopies show that little or no non-functional iron remains in the reconstituted oxyhemerythrin after the redox cycle. Quantitations of protein and diiron sites show that, during the first step of the redox cycle, the non-functional iron is converted to a form that is spectroscopically indistinguishable from that of native methemerythrin. Far-UV circular dichroism shows that the secondary structure of this reconstituted methemerythrin is essentially identical to that of native protein. Non-denaturing polyacrylamide gel electrophoresis shows that the size and charge of the native and reconstituted proteins before and after redox cycling are essentially identical. These results indicate that the non-functional iron is converted to a functional form by the redox cycling, and that the key step in this conversion is the [oxy/30% non-functional]----met transformation.
Zhang
J H
JH
Department of Chemistry, University of Georgia, Athens 30602.
Kurtz
D M
DM
Xia
Y M
YM
Debrunner
P G
PG
D006422:Hemerythrin / Q000031:analogs & derivatives / Q000737:chemistry* / Q000302:isolation & purification; D007501:Iron / Q000737:chemistry*; D008386:Marine Biology; D010084:Oxidation-Reduction; D013053:Spectrophotometry / Q000379:methods
D006422:Hemerythrin; C019004:methemerythrin; C008571:myohemerythrin; C068346:oxyhemerythrin; D007501:Iron