The binding of molybdate to uteroferrin. Hyperfine interactions of the binuclear center with 95Mo, 1H, and 2H.
Uteroferrin, an acid phosphatase with a spin-coupled and redox-active binuclear iron center, is paramagnetic in its pink, enzymatically active, mixed-valence (S = 1/2) state. Phosphate, a product and inhibitor of the enzymatic activity of uteroferrin, converts the pink, EPR-active form of the protein to a purple, EPR-silent species. In contrast, molybdate, a tetrahedral oxyanion analog of phosphate, transforms the EPR spectrum of uteroferrin from a rhombic to an axial form. With both electron spin echo envelope modulation (ESEEM) and electron nuclear double resonance (ENDOR) spectroscopies, we observe a hyperfine interaction of [95Mo]molybdate with the S = 1/2, Fe(II)-Fe(III) center of the protein. A pair of 95Mo resonances centered at the 95Mo Larmor frequency at the applied magnetic field and separated by a hyperfine coupling constant of 1.2 MHz is evident. Therefore, a single monomeric species of molybdate is close to, and likely a ligand of, the binuclear cluster. 1H ENDOR studies on uteroferrin reveal at least six sets of lines mirrored about the 1H Larmor frequency. Two pairs of these lines become reduced in intensity when the protein is exchanged against D2O. Moreover, ESEEM and 2H ENDOR spectra display resonances at the 2H Larmor frequency. Therefore, the metal-binding region of the protein is accessible to solvent. Additional deuterium lines observable by ESEEM spectroscopy provide evidence for a population of strongly coupled, readily exchangeable protons associated with the binuclear center. The measured hyperfine coupling constants for these deuterons are orientation-dependent with splittings of nearly 4 MHz at g3 = 1.59 and less than 1 MHz at g1 = 1.94. In the presence of molybdate, ESEEM spectra of D2O-exchanged samples reveal a resonance at the 2H Larmor frequency, with no evidence of spectral components due to strongly coupled deuterons. 1H ENDOR studies of the uteroferrin-molybdate complex show at least seven pairs of lines, mirrored about the 1H Larmor frequency, of which one pair becomes attenuated in amplitude upon deuteration. The active site thus remains accessible to solvent in the presence of molybdate.
Doi
K
K
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461.
McCracken
J
J
Peisach
J
J
Aisen
P
P
D000135:Acid Phosphatase; D000818:Animals; D001665:Binding Sites; D003903:Deuterium; D004578:Electron Spin Resonance Spectroscopy; D005260:Female; D006859:Hydrogen; D007527:Isoenzymes; D007554:Isotopes; D008667:Metalloproteins / Q000378:metabolism*; D008982:Molybdenum / Q000378:metabolism*; D011485:Protein Binding; D012997:Solvents; D013053:Spectrophotometry; D013552:Swine; D000071681:Tartrate-Resistant Acid Phosphatase
D007527:Isoenzymes; D007554:Isotopes; D008667:Metalloproteins; D012997:Solvents; C044659:molybdate; D006859:Hydrogen; D008982:Molybdenum; D003903:Deuterium; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase