Redox-Dependent Structural Changes in the Superoxide Reductase from Desulfoarculus baarsii and Treponema pallidum:  A FTIR Study

Abstract

The redox-induced structural changes at the active site of the superoxide reductase (SOR) from Desulfoarculus baarsii and Treponema pallidum have been monitored by means of FTIR difference spectroscopy coupled to electrochemistry. With this technique, the structure and interactions formed by individual amino acids at a redox site can be detected. The infrared data on wild-type, Glu47Ala, and Lys48Ile mutants of the SOR from D. baarsii provide experimental support for the conclusion that the two different coordination motifs observed in the three-dimensional structure of the SOR from Pyrococcus furiosus [Yeh, A. P., Hu, Y., Jenney, F. E., Adams, M. W. W., and Rees, D. (2000) Biochemistry 39, 2499-2508] correspond to the two redox forms of the SOR iron center. We extend this result to the center II iron of SOR of the desulfoferrodoxin type. Similar structural changes are also observed upon iron oxidation in the SOR of T. pallidum. In D. baarsii, the IR modes of the Glu47 side chain support that it provides a monodentate ligand to the oxidized iron, while it does not interact with Fe(2+). Structural changes at the level of peptide bond(s) observed upon iron oxidation in wild-type are suppressed in the Glu47Ala mutant. We propose that the presence of the Glu side chain plays an important role for the structural reorganization accompanying iron oxidation. We identified the infrared modes of the Lys48 side chain and found that a change in its environment occurs upon iron oxidation. The lack of other structural changes upon the Lys48Ile mutation shows that the catalytic role of Lys, as evidenced by pulse radiolysis experiments [Lombard, M., Houée-Levin, C., Touati, D., Fontecave, M., and Nivière, V. (2001) Biochemistry 40, 5032-5040], is purely electrostatic, guiding superoxide toward the reduced iron.

Long Distance Charge Redistribution Upon Cu,Zn-Superoxide Dismutase Reduction

“…This signal, assigned to a His (C 5 N ) ring mode by normal mode calculations (86), was also observed for the FeHis-4Cys superoxide reductase center of Desulfoarculus baarsii (85) and for the histidine ligands of the primary donor chlorophylls of photosystem I (83). The experiments performed here on MeIm copper or zinc complexes confirm that this mode is enhanced upon metal coordination and is a sensitive infrared marker of histidine coordination to a metal through N .…”

“…In conclusion, the long distance charge redistribution observed at the level of peptide groups upon copper reduction and the preserved electrostatic properties of the superoxide access channel may constitute a specific mechanism of Cu,Zn-SOD that contrasts to the reorganizations observed for the amino acid side chains near the iron-active site of superoxide reductase (85) and may be a key factor of the superoxide dismutase function.…”

An engineered two-iron superoxide reductase lacking the [Fe(SCys) 4 ] site retains its catalytic properties in vitro and in vivo

“…potential was very similar to that determined for the wildtype 2Fe-SOR, ϩ272 Ϯ 7 mV, under the same conditions. Midpoint reduction potentials of [Fe(NHis) 4 (SCys)] sites in SORs have been reported over the range of ϩ90 to ϩ430 mV vs. NHE (23,41), depending on the technique used, but most of the reported values cluster within the range of 200-250 mV (27, 32-34, 36, 37).…”

Geometries and Electronic Structures of Cyanide Adducts of the Non-Heme Iron Active Site of Superoxide Reductases: Vibrational and ENDOR Studies

“…In an "essentially" linear Fe-C-N complex (∼175°), where the bending mode is nonetheless allowed, the resonance Raman spectrum typically exhibits a "monotonic-isotope-shifting" ν(Fe-CN) stretching mode with a frequency 30−60 cm −1 higher than the lower energy "zigzag-isotope-shifting" δ(Fe-C-N) bending mode (30,36,38). A monotonically-isotope-shifting mode exhibits cyanide isotope shifts that vary as a function of total cyanide mass, with the 13 C 14 N and 12 C 15 N isotopomers having similar frequencies, and is characteristic of the ν(Fe-CN) stretching mode of a near-linear Fe-C-N unit A zigzag-isotope-shifting mode exhibits isotope shifts that primarily vary with isotopic substitution of the carbon atom, and thus move in a "zigzag" fashion with the isotopic sequence CN − , 13 CN, C 15 N and 13 C 15 N; this zigzag isotope shifting is characteristic of a mode primarily composed of δ(Fe-C-N) bending character. However, as the Fe-C-N angle decreases from 180°, the stretching and bending modes mix, the two frequencies move apart with lower frequency mode assuming greater ν(Fe-CN) stretching character, and both bands exhibit partially zigzag-isotope-shifting patterns depending on the extent of the mixing.…”