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Figure 39.This domain contains a slightly distorted tetrahedral [Fe(Cys)4] site that has a high homology to D. gigas desulforedoxin (Dx).364b This site, which is often referred to as "center I", was initially proposed to mediate intradomain electron transfer between a 2Fe-SOR redox partner and its SOR active center, but no evidence supporting this claim has been produced so far. In fact, the Fe- Fe distance is about 25 A, too large to allow catalytically significant intramolecular electron transfer. Instead, Emerson et al. have shown that the disruption of center I has no effect on the in vitro or in vivo activity of the D. vulgaris 2Fe-SOR.367b Additionally, the T. pallidum 1Fe-SOR, which can be viewed as a "naturally mutated" 2Fe-SOR that lost center I, retains its efficient in vitro and in vivo SOR activity.386b The ~15 residues loop connecting the two domains in the 2Fe-SORs or between the short 310 helix and the catalytic domain contains the mostly conserved (E)(K)HxP-motif.The change in conformation upon oxidation/reduction is expected to also occur in 2Fe-SORs, and indeed Fourier transform infrared (FTIR) studies suggest that the glutamate binds the Fe3+ center in the SORs from D. baarsii and T. pallidum and that this process is coupled to the change in the Fe redoxstate.385b Unfortunately, no crystallographic data have so far supported the binding of the glutamate ligand to the center in 2Fe-SORs, probably due to the difficulty of maintaining these enzymes in the Fe3+ state, in part due to X-ray-induced photoreduction of the protein during structural data collection.71b,389a,390 7.3.3.(A) Superimposition of 1Fe-SOR (blue) and 2Fe-SOR (red) monomers (PDB codes: 1DO6 and 1DFX); (B) structural conservation of amino acid residues in the monomer of 1Fe-SOR, mapped over P. furiosus structure and made using ConSurf;398,420 and (C) ribbon diagram of the same monomer in B (PDB code: 1DO6).While the SOR reduces superoxide to hydrogen peroxide, P450 binds O2 and catalyzes its two-electron reduction and double protonation to cleave the O-O bond, yielding 1 equiv of water and an enzyme intermediate known as Compound I (see section 7.5).The structure of the reduced center was determined after incubation of P. furiosus SOR crystals with sodium dithionite, showing the typical [Fe(His)4Cys] configuration (PDB code: 1DDK71b), albeit with low Fe occupancy at two of its subunits (~20%).Redox-Linked Structural Changes in the SOR Active Center.Structural conservation in SORs.7.3.2.

Figure 39. Structural conservation in SORs. (A) Superimposition of 1Fe-SOR (blue) and 2Fe-SOR (red) monomers (PDB codes: 1DO6 and
1DFX); (B) structural conservation of amino acid residues in the monomer of 1Fe-SOR, mapped over P. furiosus structure and made using
ConSurf;398,420 and (C) ribbon diagram of the same monomer in B (PDB code: 1DO6). Panels A and C were generated using the PyMOL
Molecular Graphics Systemthe immunoglobulin-like catalytic domain by a short stretch of
ca. 15 residues, having a short 310 helix. This domain contains a
slightly distorted tetrahedral [Fe(Cys)4] site that has a high
homology to D. gigas desulforedoxin (Dx).364b This site, which
is often referred to as “center I”, was initially proposed to
mediate intradomain electron transfer between a 2Fe-SOR
redox partner and its SOR active center, but no evidence
supporting this claim has been produced so far. In fact, the Fe−
Fe distance is about 25 Å, too large to allow catalytically
significant intramolecular electron transfer. Instead, Emerson et
al. have shown that the disruption of center I has no effect on
the in vitro or in vivo activity of the D. vulgaris 2Fe-SOR.367b
Additionally, the T. pallidum 1Fe-SOR, which can be viewed as
a “naturally mutated” 2Fe-SOR that lost center I, retains its
efficient in vitro and in vivo SOR activity.386b The ∼15 residues
loop connecting the two domains in the 2Fe-SORs or between
the short 310 helix and the catalytic domain contains the mostly
conserved (E)(K)HxP-motif. In both SOR types, the active
centers are very solvent exposed, and this is determinant for the
SOR catalytic features. As expected, the amino acid residues
near the catalytic center are rather conserved, while those far
from it are quite variable (Figure 39B).
The unprecedented structural features of the SOR active
center [Fe(His)4Cys] can only be compared to the active site of
cytochrome P450 monooxygenase enzymes (P450). In P450,
the Fe is also coordinated by four nitrogens from a porphyrin
ring and by an axial cysteine ligand. Despite these similarities,
the chemistries of the SOR and P450 centers are quite different.
While the SOR reduces superoxide to hydrogen peroxide, P450
binds O2 and catalyzes its two-electron reduction and double
protonation to cleave the O−O bond, yielding 1 equiv of water
and an enzyme intermediate known as Compound I (see
section 7.5).
7.3.2. Redox-Linked Structural Changes in the SOR
Active Center. When in its Fe3+ state, the SOR [Fe(His)4Cys]
center can adopt an octahedral geometry, with an extra
glutamate ligand binding to the Fe in the position opposite to
the axial cysteine [Fe(His)4CysGlu] (Figure 38A.1). This
conformation was only unambiguously shown in the crystallo￾graphic structures of the 1Fe-SORs from P. furiosus and P.
horikoshii (PDB codes: 1DO6, 1DQI,71b and 2HVB). The
structure of the reduced center was determined after incubation
of P. furiosus SOR crystals with sodium dithionite, showing the
typical [Fe(His)4Cys] configuration (PDB code: 1DDK71b),
albeit with low Fe occupancy at two of its subunits (∼20%).
Thus, the change in the Fe redox-state in 1Fe-SORs is
accompanied by a change in the Fe coordination, and this
occurs together with a significant movement of two loop
regions (Gly9-Lys15 and Gly36-Pro40, P. furiosus SOR
numbering). These structural changes are expected to occur
during catalysis, but their importance for the actual catalytic
mechanism is still not fully understood (see section 7.5). The
change in conformation upon oxidation/reduction is expected
to also occur in 2Fe-SORs, and indeed Fourier transform
infrared (FTIR) studies suggest that the glutamate binds the
Fe3+ center in the SORs from D. baarsii and T. pallidum and
that this process is coupled to the change in the Fe redoxstate.385b Unfortunately, no crystallographic data have so far
supported the binding of the glutamate ligand to the center in
2Fe-SORs, probably due to the difficulty of maintaining these
enzymes in the Fe3+ state, in part due to X-ray-induced
photoreduction of the protein during structural data
collection.71b,389a,390
7.3.3. Crystallographic Structures of Reaction Inter￾mediates. Upon superoxide reduction, an Fe3+−(hydro)-
peroxo species is formed (see catalytic mechanism below). Such
an intermediate has been trapped in D. baarsii 2Fe-SOR crystals
after incubation with H2O2, and the structure shows the
(hydro)peroxo group bound in an end-on geometry to the Fe
ion (Figure 40).370c In this elegant experiment, the presence of
the (hydro)peroxo ligand was determined by resonance Raman
measurements on the crystal. The position of the (hydro)-
peroxo in the center appears to be stabilized by the lysine
residue (Lys48 in D. baarsii) that is part of the conserved
(E)(K)HxP-motif and by hydrogen bonding to the (hydro)-
peroxo either directly or through water molecules. This lysine
residue appears also to contribute to a positively charged
antenna to attract anions into the Fe2+ center.390