ROLES OF HEME IRON-COORDINATING HISTIDINE-RESIDUES OF HUMAN HEMOPEXINEXPRESSED 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 investigatin g their participation in heme binding. The recombinant Hx proteins wer e purified by sequential chromatography on Con A-agarose and SP-Sephar ose. The purified recombinant wild-type Hx retained its heme binding. The binding constant for heme was considerably reduced, however, sugge sting that glycosylation contributes critically to the heme binding pr operty of fix. Mutation either at His-127 or at His-56 plus His-127, b ut 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 contr ibutes to the high affinity for heme. We recorded proton NMR spectra t o investigate the possibility that the degree of high-spin content is increased by deletion of an axial His-iron coordination. H-1 NMR data indicate that each of the single-mutant heme-Hx complexes is predomina ntly low-spin, perhaps owing to coordination of the heme iron by the T hr side-chain oxygen or water oxygen coordinating to the iron.