Resonance Raman Characterization of the Mononuclear Iron Active-Site Vibrations and Putative Electron Transport Pathways in Pyrococcus furiosus Superoxide Reductase

Abstract

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 (32)S with (34)S, (14)N with (15)N, (56)Fe with (54)Fe, and exchange into a H(2)(18)O 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(p(pi))-to-Fe(d(pi)) 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-C(beta)-C(alpha) 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-C(beta)-C(alpha)-N unit for the coordinated cysteine and significant (His)N(p(pi))-Fe(d(xy))-(Cys)S(p(pi)) 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 deltaN 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.

Nitric oxide binding at the mononuclear active site of reduced Pyrococcus furiosus superoxide reductase

“…Recombinant natural abundance and 34 S globally enriched P. furiosus SOR was over-expressed in Escherichia coli and purified to homogeneity according to published procedures (8,9). Unless otherwise indicated, the samples of P. furiosus SOR used in this work were in 100 mM Hepes buffer at pH 7.5 and were reduced by using 10 mM ascorbate.…”

“…Spectroscopic and crystallographic studies of the 1Fe-SOR from Pyrococcus furiosus (7)(8)(9) and the 2Fe-SOR from Desulfovibrio desulfuricans (6,10,11) have shown that the mononuclear iron active site is ligated by four equatorial histidines (3 N and 1␦N) and one axial cysteinate in a square-pyramidal arrangement. The sixth coordination site appears to be occupied by a monodentate glutamate in the oxidized state but is vacant or occupied by a weakly coordinated water molecule in the reduced state, thereby providing a site for superoxide binding and reduction.…”

Sulfur K-Edge X-ray Absorption Spectroscopy and Density Functional Theory Calculations on Superoxide Reductase: Role of the Axial Thiolate in Reactivity

“…Note that in Compound 0 of peroxidases the protonation of a proximal Oxygen of a Fe III -OOH species is calculated to have a barrier of 9 Kcal/mol (including ZPE correction). 71 However this is still less than the ΔG ± of diffusion of a small ion in solution (11)(12) Kcal/mol) involved in the formation of the Fe II -O 2 − adduct. The following step is cleavage of the weak Fe-(O 2 H 2 ) bond which is endothermic by only E = 8 Kcal/mol and this process is associated with a further 0.03 Å decrease in the Fe-S bond length.…”

“…al. 10,11 It has a high-spin ground state and an intense CysS − → Fe III charge transfer (CT) band at 15100 cm −1 (ε ~2500 M −1 cm −1 ) which gives it its characteristic blue color. The EPR spectrum of the high-spin resting state indicated that it is quite axial (E/D = 0.06) and VTVH MCD indicated that a trans N His -Fe-N His axis defined the unique direction of the ZFS tensor and not the trans S Cys -Fe-O Glu axis.…”

Intermolecular electron transfer in two-iron superoxide reductase: a putative role for the desulforedoxin center as an electron donor to the iron active site

“…It was proposed that center I could serve as an electronic relay between the cellular reductases and the catalytic iron site but the distance of 22 Å between iron center I and center II [11,14] was considered too long for efficient intramolecular electron transfer [28]. A well conserved tyrosine residue in 2Fe-SOR (Y115 in D. baarsii) located between center I and center II, at 10 Å from iron center II, was proposed to act as an electronic relay between the two iron centers ( Figure 1) [29], but up to now no studies have investigated these hypotheses.…”