Resonance Raman characterization of the mononuclear iron active-site vibrations and putative electron transport pathways in Pyrococcus furiosus superoxide reductase

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The resonance Raman spectrum of oxidized wild-type P. furiosus SOR at pH 7.5 and 10.5 has been investigated using excitation wavelengths between 406 and 676 nm, and vibrational modes have been assigned on the basis of isotope shifts resulting from global replacements of 32S with 34S, 14N with 15N, 56Fe with 54Fe, and exchange into a H 2 18O buffer. The results are interpreted in terms of the crystallographically defined active-site structure involving a six-coordinate mononuclear Fe center with four equatorial histidine ligands and axial cysteine and monodentate glutamate ligands (Yeh, A. P., Hu, Y., Jenney, F. E., Adams, M. W. W., and Rees, D. C. (2000) Biochemistry 39, 2499-2508). Excitation into the intense (Cys)S(pπ)-to-Fe(dπ) CT transition centered at 660 nm results in strong enhancement of modes at 298 cm -1 and 323 cm -1 that are assigned to extensively mixed cysteine S-Cβ-Cα bending and Fe-S(Cys) stretching modes, respectively. All other higher-energy vibrational modes are readily assigned to overtone or combination bands or to fundamentals corresponding to internal modes of the ligated cysteine. Weak enhancement of Fe-N(His) stretching modes is observed in the 200-250 cm -1 region. The enhancement of internal cysteine modes and Fe-N(His) stretching modes are a consequence of a near-planar Fe-S-Cβ-Cα-N unit for the coordinated cysteine and significant (His)N(pπ)-Fe(d xy)-(Cys)S-(pπ) orbital overlap, respectively, and have close parallels to type 1 copper proteins. By analogy with type 1 copper proteins, putative superexchange electron-transfer pathways to the mononuclear Fe active site are identified involving either the tyrosine and cysteine residues or the solvent-exposed δN histidine residue in a Y-C-X-X-H arrangement. Studies of wild-type at pH 10.5 and the E14A variant indicate that the resonance Raman spectrum is remarkably insensitive to changes in the ligand trans to cysteine and hence are inconclusive concerning the origin of the alkaline transition and the nature of sixth Fe ligand in the E14A variant.

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This article was published in Biochemistry, Volume 41, Issue 31, Pages 9833-9841.

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