Purple Uteroferrin Research Articles

1H NMR studies of porcine uteroferrin. Magnetic interactions and active site structure.

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.

Characterization of pink and purple uteroferrin by resonance Raman and CD spectroscopy.

Characterization of pink and purple uteroferrin by resonance Raman and CD spectroscopy.

EPR signal, purple color, and iron binding in porcine uteroferrin.

Titration of purple uteroferrin with two reducing equivalents of ferrous ion doubles the intensity of its g' = 1.74 EPR signal while inducing only minor changes in spectral characteristics. Unexpectedly, intensification of the EPR signal is not accompanied by a commensurate increase in visible absorption: only a small shift in peak position from 545 to 525 nm is observed. These observations suggest that uteroferrin can bind a second iron to form a paramagnetic complex that is essentially nonchromophoric. Titration of the pink protein with one oxidizing equivalent of ferric ion also doubles the intensity of its g' = 1.74 EPR signal, again shifting the primary visible absorption band to 525 nm. In either case, therefore, it is possible to augment the g' = 1.74 EPR signal without a corresponding augmentation of purple-pink color. Conversely, the addition of hydrogen peroxide (but not ferricyanide) to purple uteroferrin obliterates the g' = 1.74 EPR signal without abolishing the protein's visible absorption spectrum. Hence, it is also possible to have purple color without a corresponding g' = 1.74 EPR signal. To explain these curious results, two possible models, one involving low spin ferric iron exclusively and the other a combination of low spin ferric and high spin ferrous, are adduced.

The novel “g' = 1.74” EPR spectrum of pink and purple uteroferrin.

Low temperature (T less than or equal to 20 K) EPR measurements have revealed the presence of a heretofore undetected signal in uteroferrin, a purple protein bearing a single iron atom per molecule. All three of its principal g values (1.923, 1.738, and 1.583) lie well below the free electron value of 2.0023. Magnetic susceptibility data from 2-77 K confirm that the novel EPR spectrum arises from a paramagnetic center with a single unpaired electron spin. Quantitative correlation of the EPR, susceptibility, and optical data point to chromophoric iron as the source of the rhombic EPR spectrum. Furthermore, close agreement between the concentration of iron and the integrated intensity of the rhombic EPR signal show that the iron in the paramagnetic center is mononuclear. Reduction of the protein to its pink form leaves the rhombic signal essentially unaltered. The previously reported g' = 4.3 EPR signal accounts for only a small fraction of the total iron in the protein and undoubtedly arises from adventitious iron. Collectively, these results strongly suggest that uteroferrin represents a new class of low spin iron proteins.