Abstract
Pink (reduced) uteroferrin exhibits well resolved paramagnetic NMR spectra with resonances ranging from 90 ppm downfield to 70 ppm upfield. The intensities of these signals depend on the degree of reduction and correlate well with the intensity of the EPR signals with gave = 1.74. Analyses of chemical shifts and the temperature dependence of the paramagnetically shifted resonances indicate that the Fe(III)-Fe(II) cluster in the reduced protein exhibits weak antiferromagnetic exchange coupling (-J approximately equal to 10 cm-1), in agreement with the estimate derived from the temperature dependence of the EPR signal intensity. Purple (oxidized) uteroferrin, on the other hand, exhibits no discernible paramagnetically shifted resonances, reflecting either strong antiferromagnetic coupling or an unfavorable electron spin-lattice relaxation time. Evans susceptibility comparisons between pink and purple uteroferrin show that the Fe(III)-Fe(III) cluster in the oxidized protein is more strongly coupled (-J greater than 40 cm-1). This value concurs with low temperature magnetic susceptibility measurements on both the porcine and splenic purple acid phosphatases. The isotropically shifted protons of tyrosine coordinated to the cluster are assigned by comparison with synthetic complexes. Tyrosine, earlier implicated as a ligand by resonance Raman spectroscopy, appears to coordinate only to the ferric site in pink uteroferrin. This is consistent with the relatively invariant extinction coefficients of uteroferrin in its oxidized and reduced forms and the ease of reduction of the nonchromophoric iron compared to its chromophoric partner. Other possible ligands to the cluster include histidine, suggested by the presence of downfield-shifted solvent-exchangeable resonances with appropriate isotropic shifts.
Highlights
Pinkuteroferrinexhibits wellresolved form,X,(, -570 nm) which is enzymatically inactive or a paramagnetic NMR spectra with resonances ranging pink, reduced form,X(, -510 nm) which is enzymatically from 90 ppm downfield to 70 ppm upfield
The purple-to-pink conversion is effected by mild reductants such as P-mercaptemperature dependence of thpearamagnetically toethanol, ascorbate, and dithiothreitol, while oxidants such shifted resonances indicatethat theFe(II1)-Fe(I1)cluster in the reducedprotein exhibits weak antiferromagnetic exchange coupling(-J 9 10 cm”), in agreement with the estimate derived from the temperature dependence of the EPR signal intensity
Our data provide: further insights into the ligand by resonance Raman spectroscopy, appears to nature of the two oxidation forms of uteroferrin; an estimate coordinate only to the ferric site in pink uteroferrin. for the strength of the antiferromagnetic interaction in both. This is consistent with the relatively invariant extinco- xidized and reduced forms; and evidence for the presence of tion coefficients of uteroferrin in itsoxidized and re- tyrosine and histidine in the coordination of the binuclear duced forms and theaese of reduction of the nonchromophoric iron compared to its chromophoric partner
Summary
Evidence for a uteroferrin show that theFe(II1)-Fe(II1)cluster in the binuclear site has recently been reported for uteroferrin (11, oxidized protein is more strongly coupled (-J > 40 14) and for the beef spleen purple acid phosphatase which cm”). This value concurs withlow temperature mag- exhibits many of the physicochemicalproperties of uteroferrin netic susceptibility measurements on boththe porcine [7, 15,16,17]. Samples reduced to different degrees were obtained by varying the time of exposure to reductant
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