Electron Spin-echo Envelope Modulation Spectroscopy Research Articles

Electron spin-echo spectroscopic studies of Escherichia coli fumarate reductase.

Electron spin-echo envelope modulation (ESEEM) spectroscopy was applied to the study of reduced Centre 1 of Escherichia coli fumarate reductase (succinate:(acceptor) oxidoreductase, EC 1.3.99.1). The ESEEM spectrum derived from stimulated (3-pulse) echo envelopes obtained at 8.8 GHz contained lines at 0.9, 2.1, 3.0 and 4.2 MHz in the g = 1.94 region. When studied at 11.4 GHz, these low-frequency components scale with magnetic field in a manner indicating interaction between the unpaired electron spin of the Fe-S cluster and a weakly coupled 14N nucleus. Spectral simulations of these ESEEM data yield nuclear quadrupole interaction parameters indicative of peptide nitrogen. For oxidized protein, the magnetic-field dependence of the linear electric-field effect (LEFE) for Centre 3 was measured, and the results confirm the presence of a [3Fe-4S] cluster in the protein.

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.

Identification of nonprotein ligands to the metal ions bound to glutamine synthetase.

Electron paramagnetic resonance (EPR) was used to study the environment of Mn2+ bound to the tight (n1) metal ion binding site of glutamine synthetase in the presence of analogues of the tetrahedral adduct, L-methionine (S)-sulfoximine [Met(O)(NH)-S] and L-methionine (R)-sulfoximine [Met(O)(NH)-R]. The Mn2+ EPR spectrum in the presence of Met(O)(NH)-S is identical with the previously published spectrum obtained from a mixture of isomers [Met(O)(NH)-RS] [Villafranca, J. J., Ash, D. E., & Wedler, F. C. (1976) Biochemistry 15, 544] and is characteristic of a highly octahedral metal ion environment with a small zero field splitting. The presence of Met(O)(NH)-R produces an EPR spectrum that appears characteristic of a more distorted metal ion environment, with a larger zero field splitting. These data demonstrate that the two isomers interact differently with the enzyme-bound Mn2+. Broadening of the Mn2+ EPR spectrum in the presence of Met(O)(NH) is observed in 17O-enriched water due to superhyperfine coupling of water to the metal ion. Deconvolution of the spectrum demonstrates the presence of at least a single water molecule in the inner coordination sphere of the metal ion. Superhyperfine coupling due to the 14N nucleus of the imine nitrogen of the sulfoximine moiety of Met(O)(NH)-S but not of Met(O)(NH)-R has been detected by electron spin-echo envelope modulation spectroscopy. Two intense peaks are evident in the presence of Met(O)(NH)-S with frequencies at 1.7 and 3.3 MHz. These peaks are absent when [15N]imine-labeled Met(O)(NH) is used, indicating the presence of the sulfoximine nitrogen of Met(O)(NH)-S in the inner coordination sphere of the metal ion.(ABSTRACT TRUNCATED AT 250 WORDS)