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 proteolytic cleavage of intact hemopexin, was studied by EPR, MCD and 1H-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. 10, 123-128) and are inconsistent with recently reported mutagenesis studies indicating that bis-histidine ligation is unlikely (Satoh et al. (1994) Proc. Natl. Acad. Sci. USA 91, 8423-8427). Although the MCD data are consistent with both bis-histidine and histidine/lysine ligation, 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 1H-NMR studies indicate that heme binding to domain I perturbs at least 4 of the 5 histidines. This is consistent with axial ligation of the heme by two histidines, and a conformational change induced by heme binding affecting two more. Interestingly, resonances of the carbohydrate bound to intact hemopexin and domain I were also perturbed by heme binding. pH dependence studies showed 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 heme.
Cox
M C
MC
Centre for Metalloprotein Spectroscopy and Biology, School of Chemical Sciences, University of East Anglia, Norwich, UK.
Le Brun
N
N
Thomson
A J
AJ
Smith
A
A
Morgan
W T
WT
Moore
G R
GR
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000737:chemistry; D001665:Binding Sites; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry*; D006466:Hemopexin / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D011817:Rabbits
D001059:Apoproteins; D008024:Ligands; D010446:Peptide Fragments; D006418:Heme; D006639:Histidine; D006466:Hemopexin