Circular Dichroism and Magnetic Circular Dichroism Studies of the Mixed-Valence Binuclear Non-Heme Iron Active Site in Uteroferrin and Its Anion Complexes

Abstract

Uteroferrin (Uf) is a purple acid phosphatase for which the reduced [Fe II Fe III ] form of the enzyme is catalytically active in the hydrolysis of phosphate esters. The binuclear non-heme iron active site of the native reduced enzyme (1/2metUf) and its molybdate-and phosphate-bound forms have been studied with absorption (ABS), circular dichorism (CD), and magnetic circular dichroism (MCD) spectroscopies to probe their geometric and electronic structure. CD and MCD in the UV-Vis region give the tyrosine (phenolate) to Fe III charge transfer bands which probe the ferric center. CD and MCD in the NIR region probe the d-d transitions of both the Fe II and the Fe III centers. Variable-temperature variable-field (VTVH) MCD combined with EPR data are analyzed to determine the g values and energies of the ground state and the excited sublevels. These parameters are further interpreted in terms of a spin Hamiltonian model, which includes the zero-field splitting (ZFS) of Fe II and Fe III centers and the exchange coupling (J) between the irons due to bridging ligation. These ground and excited-state results confirm that both irons are six-coordinate with a µ-OH bridge. Anions bind to the active site in a bridging mode, which perturbs the ground and excited states of both iron centers. In particular, the exchange coupling decreases, µ-OH bridge f Fe II bonding strength increases, and the Tyr f Fe III donor interaction increases upon the phosphate binding. These results are correlated with the recent X-ray crystal structure of the kidney bean purple acid phosphatase, which contains an Fe III Zn II active site. These studies provide geometric and electronic structure insight into the hydrolysis reaction mechanism of this enzyme.

Phosphate ester cleavage promoted by a tetrameric iron(III) complex

“…The enzyme isolated from mammals (pig, bovine, mouse, rat and human) is an approximately 35 kDa monomeric protein with an Fe(III)-Fe(II) centre, and a characteristic purple colour due to a charge-transfer transition (k max = 510-560 nm; e = *3,000-4,000 M -1 cm -1 ) from a conserved tyrosine ligand to the ferric ion ( Fig. 1) [3][4][5]. Mammalian PAPs are easily and reversibly oxidized to the inactive diferric form owing to the low redox potential (approximately 340 mV) of the divalent iron [6,7], suggesting that the PAP activity may be regulated in vivo by reversible oxidation/reduction of the active site.…”

Metal-Ion Mutagenesis: Conversion of a Purple Acid Phosphatase from Sweet Potato to a Neutral Phosphatase with the Formation of an Unprecedented Catalytically Competent MnIIMnII Active Site

“…13 The characteristic purple color of PAPs is due to the formation of a charge-transfer (CT) complex between the trivalent iron and a conserved tyrosine ligand. 14 Fe III Fe III/II center, where only the mixed-valent form is believed to be catalytically competent. 15 Reduction of the active Fe III Fe II center to Fe II Fe II leads to inactivation and loss of one or both metal ions depending upon exposure times to the reductant.…”

Inhibition studies of purple acid phosphatases: implications for the catalytic mechanism

“…1,2 The characteristic purple colour of these enzymes is due to a charge transfer transition between a ligand (tyrosinate) donor orbital and an acceptor orbital on the ferric ion. 1,3,4 PAPs catalyse the hydrolysis of a variety of phosphorylated substrates at acidic to neutral pH, and have been identified in, and characterised from animal, plant and fungal sources. [5][6][7][8] The enzyme extracted from mammalian organisms is a 35 kDa monomer and contains a redox-active Fe III -Fe III/II centre.…”