MCD, EPR AND NMR SPECTROSCOPIC STUDIES OF RABBIT HEMOPEXIN AND ITS HEME-BINDING DOMAIN

Heme binding to rabbit hemopexin and its domain I, obtained by proteol ytic cleavage of intact hemopexin, was studied by EPR, MCD and H-1-NMR spectroscopies. The data obtained support the proposal that the heme Fe(III) is coordinated by two histidine ligands (Morgan et al. (1988) J. Biol. Chem. 263, 8220-8225; Muster et al. (1991) J. Protein Chem. 1 0, 123-128) and are inconsistent with recently reported mutagenesis st udies indicating that bis-histidine ligation is unlikely (Satoh et al. (1994) Proc. Natl. Acad. Sci. USA 91, 8423-8427). Although the MCD da ta are consistent with both bis-histidine and histidine/lysine ligatio n, the EPR spectra are typical of bis-histidine ligation. Overall the magneto-optical spectra are characteristic for bis-histidine ligation. The EPR and NMR data indicate that there is a difference in the heme environments of the intact hemopexin and its domain I but overall the spectroscopic information suggests heme bound to domain I has the same ligands as intact hemopexin. The H-1-NMR studies indicate that heme b inding to domain I perturbs at least 4 of the 5 histidines. This is co nsistent with axial ligation of the heme by two histidines, and a conf ormational change induced by heme binding affecting two more. Interest ingly, resonances of the carbohydrate bound to intact hemopexin and do main I were also perturbed by heme binding, pH dependence studies show ed that heme remained bound to intact hemopexin over the pH range 6.5- 10.0 without any major change in the ligation or environment of the he me.