Magnetic Circular Dichroism and Electron Paramagnetic Resonance Studies of Cobalt-Substituted Horse Liver Alcohol Dehydrogenase

Abstract

The ground and excited state properties of Co(II) substituted for Zn(II) at the catalytic (c) and the noncatalytic (n) sites of horse liver alcohol dehydrogenase EE isozyme have been investigated by parallel EPR and UV/ visible variable-temperature magnetic circular dichroism (VTMCD) spectroscopies. Samples were investigated as prepared and after formation of a ternary complex with NAD+ and the potent inhibitor pyrazole. In accord with the structural role proposed for the noncatalytic metal, the spectroscopic properties of Co(II) at the noncatalytic site were unperturbed by formation of the ternary complex. The EPR spectra were readily analyzed in terms of a S = 3/2 spin Hamiltonian using anisotropic intrinsic g-values in the range characteristic of tetrahedral Co(II), i.e. g = 2.1-2.4; E/D 0), and 0 (with D > 0) for Co(c)Zn(n)-HLADH, Co(c)Zn(n)-HLADH/NAD+/pyrazole, and Zn(c)Co(n)-HLADH, respectively. VTMCD studies facilitated resolution and assignment of S -Co(II) charge transfer bands (300-400 nm) and the components of the 4A2 -'Tl(P) tetrahedral d-d band (500-800 nm) that are split by spin-orbit coupling and low-symmetry distortions. The splittings of the highest energy d-d band are indicative of a much more distorted coordination environment for Co(I1) at the catalytic site than the noncatalytic site. This is also reflected in the magnitude of ground state zero-field splitting, A, determined by analysis of the temperature dependence of discrete MCD bands, IA( = 33, 56, and 7 cm-* for Co(c)Zn(n)-HLADH, Co(c)Zn(n)-HLADH/NAD+/pyrazole, and Zn(c)Co(n)-HLADH, respectively. MCD magnetization data are rationalized in terms of the EPR-determined ground state effective g-values, ground state zero-field splitting, and the polarization of the electronic transitions. The zero-field splittings for the samples with Co(I1) at the catalytic site determined by VTMCD are quite different from those determined by EPR from the temperature dependence of the spin relaxation (Makinen, M. W.; Yim, M. B. Proc. Nutl. Acad. Sci. U S A . 1981 78, 6221-6225), and the origin of this discrepancy is discussed. In accord with X-ray crystallographic studies, the EPR and VTMCD data are rationalized in terms of a highly distorted tetrahedral coordination environment for Co(I1) at the catalytic site (two cysteines, one histidine, and one H20 for Co(c)Zn(n)-HLADH and two cysteines, one histidine and one pyrazole for Co(c)Zn(n)-HLADWNAD+/pyrazole) and a more regular tetrahedral environment for Co(I1) at the noncatalytic site (four cysteines). 0.33, 0.05 (with D

Reductive activation of hexavalent chromium by human lung epithelial cells: Generation of Cr(V) and Cr(V)-thiol species

“…Thus there are two Cr(III) signals in cells: the g eff = 5.2 just described and the second with g eff = 4.3 which has an E/D of about 0.04. These cellular signals are similar to the signals for the Co(II) sites (S = 3/2) in horse liver alcohol dehydrogenase [52].…”

Synthesis, Solid‐State Structures, and EPR Spectroscopic Studies on Polycrystalline and Single‐Crystal Samples of α‐Diimine Cobalt(II) Complexes

“…An effective Hamiltonian within the lowest SЈ = 1/2 (corresponding to either the originals |3/2, ±3/2Ͼ or the |3/2, ±1/2Ͼ states) Kramers doublet can be obtained under the form [31][32][33][34] H eff = µ B BgЈSЈ…”

High-spin Co(II) in monomeric and exchange coupled oligomeric structures: Magnetic and magnetic circular dichroism investigations

“…Naturally occurring cobalt metalloenzymes are not so common as iron metalloenzymes, but cobalt can replace other metal ions in some native metalloenzymes with partial or full retention of their activity, as, e.g., Zn(II) in carbonic anhydrase [14], in horse liver alcohol dehydrogenase [15] or in insulin hexamer [16]. By substituting Cu, binuclear Co centers can be formed as in the case of cobalt hemocyanin [17,18] and can be investigated by MCD spectroscopy [18].…”

“…Whereas in Ref. [15] VTVH-MCD was used as completion of the conventional EPR study, in Ref. [20a] HFEPR was compared with MCD and the results obtained from both methods were in a good agreement.…”

“…However, the evaluation of ZFS from these EPR measurements is not easy. Three resolved signals on the spectrum are needed, corresponding to effective gfactors: g x , g y , g z , from which one can calculate E/D value (see one example [15]) and use some formula to extract E and D; here E is the rhombic zero field splitting and D the axial one. One other limitation is due to the fact that the splitting energy is sometimes greater than the microwave energy, in particular by using X-band spectrometer.…”