Spectroscopic studies of Pyrococcus furiosus superoxide reductase: Implications for active-site structures and the catalytic mechanism

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The combination of UV/visible/NIR absorption, CD and variable-temperature magnetic circular dichroism (VTMCD), EPR, and X-ray absorption (XAS) spectroscopies has been used to investigate the electronic and structural properties of the oxidized and reduced forms of Pyrococcus furiosus superoxide reductase (SOR) as a function of pH and exogenous ligand binding. XAS shows that the mononuclear ferric center in the oxidized enzyme is very susceptible to photoreduction in the X-ray beam. This observation facilitates interpretation of ground- and excited-state electronic properties and the EXAFS results for the oxidized enzyme in terms of the published X-ray crystallographic data (Yeh, A. P.; Hu, Y.; Jenney, F. E.; Adams, M. W. W.; Rees, D. C. Biochemistry 2000, 39, 2499-2508). In the oxidized state, the mononuclear ferric active site has octahedral coordination with four equatorial histidyl ligands and axial cysteinate and monodentate glutamate ligands. Fe EXAFS are best fit by one Fe-S at 2.36 Å and five Fe-N/O at an average distance of 2.12 Å. The EPR-determined spin Hamiltonian parameters for the high-spin (S = 5/2) ferric site in the resting enzyme, D = -0.50 ± 0.05 cm -1 and E/D = 0.06, are consistent with tetragonally compressed octahedral coordination geometry. UV/visible absorption and VTMCD studies facilitate resolution and assignment of πHis → Fe 3+(t 2g) and (Cys)S(p) → Fe 3+(t 2g) charge-transfer transitions, and the polarizations deduced from MCD saturation magnetization studies indicate that the zero-field splitting (compression) axis corresponds to one of the axes with trans-histidyl ligands. EPR and VTMCD studies provide evidence of azide, ferrocyanide, hydroxide, and cyanide binding via displacement of the glutamate ligand. For azide, ferrocyanide, and hydroxide, ligand binding occurs with retention of the high-spin (S = 5/2) ground state (E/D = 0.27 and D 7400 cm -1. Analysis of MCD saturation magnetization data leads to ground-state zero-field splitting parameters for the S = 2 ground state, D ∼+10 cm -1 and E/D ∼ 0.1, and complete assessment of ferrous d-orbital splitting. Azide binds weakly at the vacant coordination site of reduced SOR to give a coordination geometry intermediate between octahedral and square pyramidal with 10Dq = 9700 cm -1 and Δ 5E g = 4800 cm -1. Cyanide binding results in an octahedral ferrous site with 10Dq = 10 900 cm -1 and Δ 5E g = 1750 cm -1. The ability to bind exogenous ligands to both the ferrous and ferric sites of SOR is consistent with an inner-sphere catalytic mechanism involving superoxide binding at the ferrous site to yield a ferric-(hydro)peroxo intermediate. The structural and electronic properties of the SOR active site are discussed in relation to the role and bonding of the axial cysteine residue and the recent proposals for the catalytic mechanism.

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Journal of the American Chemical Society





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This article was published in Journal of the American Chemical Society, Volume 124, Issue 5, Pages 788-805.

The published version is available at http://dx.doi.org/10.1021/ja016889g.

Copyright © 2002 ACS.

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