Magnetization and electron paramagnetic resonance studies of reduced uteroferrin and its "EPR-silent" phosphate complex.

Spectroscopic and Enzymatic Characterization of the Active Site Dinuclear Metal Center of Calcineurin: Implications for a Mechanistic Role

“…Phosphate addition to the Fe 3+ -Fe 2+ form of calcineurin, on the other hand, led to a decrease in intensity and significant broadening of the signal from the mixed valence center. In fact, the EPR relaxation properties of the phosphate complex of calcineurin, such as a tolerance to power saturation at low temperature and an increased sensitivity to temperature, are similar to those observed for the uteroferrin‚phosphate complex (Day et al, 1988). With uteroferrin‚phosphate, the perturbations of the EPR signal of the mixed valence cluster were explained to result from a decrease in the exchange coupling constant J for the mixed valence cluster, possibly by protonation of the bridging solvent molecule of the cluster.…”

“…Recent crystallographic studies of the tungstate complex of PP1 (Egloff et al, 1995), the phosphate complex of calcineurin (Griffith et al, 1995), and the phosphate and tungstate complexes of kidney bean purple acid phosphatase (Klabunde et al, 1996) suggest that inhibition occurs by bidentate coordination of both metal ions of the cluster. EPR and susceptibility (Day et al, 1988), Mo ¨ssbauer (Pyrz et al, 1986, and EXAFS (Wang et al, 1996b) studies of uteroferrin are also consistent with this mode of inhibition. The addition of phosphate to both the Fe 3+ -Zn 2+ and Fe 3+ -Fe 2+ forms of calcineurin led to distinct changes in the corresponding EPR signals.…”

Evidence for Nonbridged Coordination of p-Nitrophenyl Phosphate to the Dinuclear Fe(III)−M(II) Center in Bovine Spleen Purple Acid Phosphatase during Enzymatic Turnover

“…a Obtained from a fit of kcat as a function of pH using eq 1. b Obtained from a fit of kcat/KM as a function of pH using eq 2, with pKe1 < 2. Fe(III)Fe(II)-Uf‚PO 4 , which showed g-values at 2.25, 1.5, and ∼1.1 (8). The feature at g ) 1.52 seems to also be present in the EPR spectrum of the uncomplexed Fe(III)Fe-(II)-BSPAP, and may be due to a small amount of the protein that is not able to bind phosphate.…”

Binuclear Metallohydrolases

“…Mössbauer measurements identified two distinct low-symmetry 6-coordinate high-spin Fe(III) ions in the oxidized form and a mixed-valence site, consisting of high-spin Fe(III) and high-spin Fe(II), upon reduction to the active form. , The electronic structure of the Fe(III)Fe(II) PAP site has now been characterized in considerable detail in a recent Mössbauer study . Magnetic susceptibility, EPR, and NMR measurements have indicated a strong antiferromagnetic interaction in the oxidized enzyme (−2 J > 80 cm -1 ) ,,,, but a weaker antiferromagnetic coupling in the reduced enzyme (2 J = −11 to −22 cm -1 ). − 1 H NMR data have been particularly informative about the ligands, initially indicating His coordination and identifying Tyr as a Fe(III) ligand in the reduced enzyme . NMR measurements later provided evidence for Nε coordination of His to the Fe(III) and Nδ coordination of Histo the Fe(II) in the reduced enzyme and for carboxylate coordination to the metal ions. − Advanced EPR techniques, including ENDOR, ESEEM, , and LEFE, have confirmed His ligation and a trapped valence description of the Fe(III)Fe(II) site in the reduced enzyme and have demonstrated solvent accessibility and probable coordination to the metal ions.…”