Edmund P. Day

Spectroscopic and magnetochemical studies on the active site copper complex in galactose oxidase

Abstract Galactose oxidase is a radical copper oxidase, an enzyme making use of a covalently modified tyrosine residue as a free radical redox cofactor in alcohol oxidation catalysis. We report here a combination of spectroscopic and magnetochemical studies developing insight into the interactions between the active site Cu(II) and two distinct tyrosine ligands in the biological complex. One of the tyrosine ligands (Y495) is coordinated to the Cu(II) metal center as a phenolate in the resting enzyme and serves as a general base to abstract a proton from the coordinated substrate, thus activating it for oxidation. The structure of the resting enzyme is temperature-dependent as a consequence of an internal proton equilibrium associated with this tyrosine that mimics this catalytic proton transfer step. The other tyrosine ligand (Y272) is covalently crosslinked to a cysteine residue forming a tyrosine–cysteine dimer free radical redox site that is required for hydrogen atom abstraction from the activated substrate alkoxide. The presence of the free radical in the oxidized active enzyme results in formation of an EPR-silent Cu(II) complex shown by multifield magnetic saturation experiments to be a diamagnetic singlet arising from antiferromagnetic exchange coupling between the metal and radical spins. A paramagnetic contribution observed at higher temperature may be associated with thermal population of the triplet state, thus permitting an estimate of the magnitude of the isotropic exchange coupling ( J >200 cm −1 , JS 1 · S 2 ) in this complex. Structural correlations and the possible mechanistic significance of metal–radical coupling in the active enzyme are discussed.

Magnetization of manganese superoxide dismutase from Thermus thermophilus

The ground state magnetic properties of manganese superoxide dismutase from Thermus thermophilus in its native and reduced forms have been determined using saturation magnetization data. Parallel EPR measurements were used to verify that commonly encountered paramagnetic impurities were at low concentration relative to the metalloprotein. The native enzyme contains high spin Mn(III) (S = 2) with D = +2.44(5) cm-1 and E/D = 0. The reduced enzyme contains high spin Mn(II) (S = 5/2) with D = +0.50(5) cm-1 and E/D = 0.027. These results are in keeping with the suggestions of several previous groups of workers concerning the permissible oxidation and spin states of the manganese, but the zero field splitting parameters are unlike those of known manganese model compounds. In addition, the extinction coefficient for the visible region absorption maximum of the native enzyme and the corresponding difference extinction coefficient (native minus reduced) have been measured using saturation magnetization data to quantitate Mn(III) present. The result, epsilon 480 = 950(80) M-1 cm-1 (delta epsilon 480 = 740(60) M-1 cm-1) agrees with the previously reported value of epsilon 480 = 910 M-1 cm-1 found by total manganese determination (Sato, S. and Nakazawa, K. (1978) J. Biochem. 83, 1165-1171). The wide variation in the reported visible region extinction coefficients of manganese superoxide dismutases from different sources is discussed.

Nuclear quadrupole double resonance spectrometer with magnetic property measurement system direct current superconducting quantum interference device detector and automatic tuning

A new nuclear quadrupole double resonance spectrometer based on a commercial superconducting quantum interference device (SQUID) setup (a Magnetic Property Measurement System (MPMS) from Quantum Design) is described. The experiment involves the indirect detection of pure quadrupole resonances (PQR) of a dilute spin system via the direct SQUID detected NMR of an abundant spin system. The experiment is conducted at low (3-20 K) temperatures and the magnetic field is cycled between a high (5.5 T) polarizing field, to an intermediate (0.1 T) detection field, to zero field where the sample is irradiated with a modulated search RF and back to the detection field. Loss of the NMR signal indicates the detection of a PQR. The RF circuit used for both the NMR and zero field irradiation is digitally controlled. Use of the External Device Control allows for the complete automation of the system. Test measurements on diphenyl ether are in good agreement with previously reported results. Pure (17)O quadrupole resonances were detected for spin systems with concentrations as low as 120 μM.

Dioxygen Reactivity Models for Cytochrome C Oxidase: Synthesis and Characterization of Oxo and Hydroxo-Bridged Porphyrin-Iron/Copper Dinuclear Complexes

The synthesis of appropriate transition-metal complexes to model the structural, spectroscopic, and magnetic properties of a metalloprotein active-site provides an opportunity to consider the function and associated mechanism of that metalloprotein at the molecular level. One nice example is the dinuclear cuprous amine-bis-pyridyl complex, which effects arene hydroxylation (albeit of the ligand m-xylyl spacer) using molecular oxygen (O2).1 This extraordinary reaction involves cleavage of the O-O bond and subsequent insertion of an oxygen atom into an arene C-H bond under essentially ambient conditions, to model the function of copper monooxygenases such as tyrosinase. Another excellent example is the generation of dicupric trans-µ-1,2-peroxo complexes from cuprous precursors and O2, reversibly,2–4 to model the oxygen-transport property of the protein hemocyanin, which subsequently was discovered to bind O2 in η2:η2 fashion, as shown in Figure 1.4 The metalloprotein cytochrome c oxidase,5 however, due to its combination of diverse and unusual active-site metal centers, has eluded a convincing model description. As for its function, it probably binds O2 at a dinuclear site comprising heme-iron and histidyl-copper coordination; it then cleaves the O-O bond, via reduction, (vide infra ).5 The structural changes associated with this dinuclear site during turnover, and the intermediates produced therefrom, are by no means clearly understood. In the resting state, the dinuclear site exhibits strong antiferromagnetic coupling (-J =200 cm-1) suggesting the involvement of a bridging ligand, often postulated as µ-sulfido, µ-chloro, or µ-hydroxo. Thus, we have endeavored to synthesize model complexes of this enigmatic dinuclear site.