Roles of heme iron-coordinating histidine residues of human hemopexin expressed in baculovirus-infected insect cells.
Hemopexin (Hx), the major heme-binding plasma glycoprotein, scavenges circulating heme and performs an antioxidant function. In the present study, human Hx was expressed in a baculovirus system and its presumed essential His residues were mutated to Thr as a means of investigating their participation in heme binding. The recombinant Hx proteins were purified by sequential chromatography on Con A-agarose and SP-Sepharose. The purified recombinant wild-type Hx retained its heme binding. The binding constant for heme was considerably reduced, however, suggesting that glycosylation contributes critically to the heme binding property of Hx. Mutation either at His-127 or at His-56 plus His-127, but not at His-56 per se, reduced the affinity for heme by an order of magnitude relative to wild-type Hx. It is concluded that His-127 contributes to the high affinity for heme. We recorded proton NMR spectra to investigate the possibility that the degree of high-spin content is increased by deletion of an axial His-iron coordination. 1H NMR data indicate that each of the single-mutant heme-Hx complexes is predominantly low-spin, perhaps owing to coordination of the heme iron by the Thr side-chain oxygen or water oxygen coordinating to the iron.
Satoh
T
T
Department of Pediatrics, Cornell University Medical College, New York, NY 10021.
Satoh
H
H
Iwahara
S
S
Hrkal
Z
Z
Peyton
D H
DH
Muller-Eberhard
U
U
D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D006418:Heme / Q000378:metabolism*; D006466:Hemopexin / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009036:Moths; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011993:Recombinant Fusion Proteins; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship
D011993:Recombinant Fusion Proteins; D006418:Heme; D006639:Histidine; D006466:Hemopexin