Steady-state kinetic studies of superoxide dismutases: properties of the iron containing protein from Escherichia coli

Synthesis, X-Ray Crystallographic Characterization, and Electronic Structure Studies of a Di-Azide Iron(III) Complex: Implications for the Azide Adducts of Iron(III) Superoxide Dismutase

“…By analogy, it would thus be anticipated that two substrate molecules could simultaneously bind to the active site under turnover conditions. 4,5,[58][59][60][61][62][63] However, other evidence suggests that only one azide can actually bind to the Fe 3+ center in Fe 3+ SOD. First, a single azide ligand (Fe-N(azide) bond length of 2.12 Å) is observed in the X-ray crystal structure of N 3 −Fe 3+ SOD (Figure 1, right), even though a high concentration of azide (100 mM NaN 3 ) was used in the corresponding mother liquor solution.…”

“…13 Our data are thus consistent with the proposal that the temperature-induced conversion from the yellow to the pink N 3 −Fe 3+ SOD complex is caused by the binding of an azide ion to the putative outer-sphere substrate prebinding site, which could perturb the Fe-N(azide) bonding interaction involving the azide ligand that is already present in the yellow species via the conserved active-site hydrogen-bond network. Independent evidence for the existence of an outer-sphere prebinding site in FeSOD has been obtained in azide-binding studies of Fe 3+ SOD at room temperature 5,65,66 as well as NO-binding studies of Fe 2+ SOD at cryogenic temperatures. 67 As discussed above, Abs and MCD spectroscopic data alone do not permit an unambiguous determination of the number of azide ligands in the LFe(N 3 ) 2 complex due to the strong mixing between the azide 1 and azide 2 π nb → Fe 3+ CT transitions.…”

“…70 Consequently, neither in FeSOD nor in MnSOD does azide actually displace any of the original active-site ligands, which implies that (i) the coordinated solvent and the Asp ligand are likely to play a crucial role in tuning the redox potential of the active site metal ion in the key reaction intermediates and (ii) a single substrate molecule can coordinate to the metal ion under turnover conditions. 4,5,[58][59][60][61][62][63] …”

“…1,2 Mn-and FeSODs, like all other SODs, disproportionate the superoxide radical anion into dioxygen and hydrogen peroxide in a two-step ping-pong-type mechanism (eqs 1a and 1b, where M = Fe or Mn) during which the metal ion cycles between the 2+ and 3+ oxidation states. [3][4][5] While structurally unrelated to the Cu/Zn-and NiSODs, Mn-and FeSODs possess nearly identical protein folds and highly homologous active sites. 6 In the resting states of both proteins, the metal ions are in five-coordinate, distorted trigonal bipyramidal ligand environments with a histidine (His) and a solvent molecule in the axial positions and two His residues and an aspartate (Asp) in the equatorial plane (Figure 1, left).…”

Novel Insights into the Basis for Escherichia coli Superoxide Dismutase's Metal Ion Specificity from Mn-Substituted FeSOD and Its Very High Em

“…A very low pK ox will tend to decrease the E m whereas a low pK red will raise E m much less since K red is smaller than [H + ] [eq 2, where K ox and K red are the acid dissociation constants of the oxidized and reduced states, respectively, E AH is the reduction potential of the fully protonated species, E m is the potential observed at a given pH, and pK ) -log(K)]. 6 In addition, protonation 5 The different active site conformation observed in the Fe(Mn)-SOD crystal structure can most simply be ascribed to the fact that this structure was obtained at a pH well above the pK ascribed to OHbinding (1) whereas the structure of MnSOD was determined at a pH well below the pK ascribed to OH -binding (53 (31,43,49), and the titrations should properly be considered to be in quasi-equilibrium. Thus, the SODs' Ems may incorporate substantial uncertainty despite our best efforts.…”