Abstract:Iron‐containing superoxide dismutases (FeSOD) are generally dimers of identical 21‐kDa monomers, each of which contains a single active site. Each active site binds one Fe ion with roughly trigonal bipyramidal geometry, employing two His and an Asp
−
residue as equatorial ligands, and one more His and a coordinated solvent as axial ligands. In the course of the catalytic cycle, the Fe alternates between the +3 and +2 states, and the coordinated solvent is believed to alternate between O… Show more
“…Another possible structural change in the binuclear centre of NOR in the catalytic turnover would be dissociation of one of the ligands (His258, His259 or Glu211) from Fe B , thus opening space for accommodation of a second NO molecule in the binuclear centre. Non-haem irons having two His and one Glu/Asp ligands are generally observed in nature, in which one of the coordination sites is occupied by a water molecule [55][56][57]. In addition, in the crystal structure of quercetin 2,3-dioxygenase, which contains copper Cu having 3 His and 1 Glu, both the Glu-bound and unbound forms are concomitantly observed in the Cu 2þ state [58,59].…”
Section: Molecular Mechanism Of No Reduction By Nor (A) Reaction Mechmentioning
The crystal structure of the bacterial nitric oxide reductase (cNOR) from
Pseudomonas aeruginosa
is reported. Its overall structure is similar to those of the main subunit of aerobic and micro-aerobic cytochrome oxidases (COXs), in agreement with the hypothesis that all these enzymes are members of the haem-copper oxidase superfamily. However, substantial structural differences between cNOR and COX are observed in the catalytic centre and the delivery pathway of the catalytic protons, which should be reflected in functional differences between these respiratory enzymes. On the basis of the cNOR structure, we propose a possible reaction mechanism of nitric oxide reduction to nitrous oxide as a working hypothesis.
“…Another possible structural change in the binuclear centre of NOR in the catalytic turnover would be dissociation of one of the ligands (His258, His259 or Glu211) from Fe B , thus opening space for accommodation of a second NO molecule in the binuclear centre. Non-haem irons having two His and one Glu/Asp ligands are generally observed in nature, in which one of the coordination sites is occupied by a water molecule [55][56][57]. In addition, in the crystal structure of quercetin 2,3-dioxygenase, which contains copper Cu having 3 His and 1 Glu, both the Glu-bound and unbound forms are concomitantly observed in the Cu 2þ state [58,59].…”
Section: Molecular Mechanism Of No Reduction By Nor (A) Reaction Mechmentioning
The crystal structure of the bacterial nitric oxide reductase (cNOR) from
Pseudomonas aeruginosa
is reported. Its overall structure is similar to those of the main subunit of aerobic and micro-aerobic cytochrome oxidases (COXs), in agreement with the hypothesis that all these enzymes are members of the haem-copper oxidase superfamily. However, substantial structural differences between cNOR and COX are observed in the catalytic centre and the delivery pathway of the catalytic protons, which should be reflected in functional differences between these respiratory enzymes. On the basis of the cNOR structure, we propose a possible reaction mechanism of nitric oxide reduction to nitrous oxide as a working hypothesis.
“…These and other mutations of the active site Gln also significantly alter the E m (10,22,25,26), consistent with our proposal that the H-bond between Gln69 and coordinated solvent exerts an important influence on the relative stabilities of coordinated H 2 O vs OH -and, thereby, the E m (1,10,22). Since proton transfer coupled to electron transfer is the norm in biological chemistry, we proposed that proteins' exquisite and strong control over the pKs and degrees of protonation of functionalities coupled to the redox sites, can provide a general and very important mechanism of redox tuning in enzymes (27,57).…”
Fe-containing superoxide dismutase's active site Fe is coordinated by a solvent molecule, whose protonation state is coupled to the Fe oxidation state. Thus, we have proposed that H-bonding between glutamine 69 and this solvent molecule can strongly influence the redox activity of the Fe in superoxide dismutase (SOD). We show here that mutation of this Gln to His subtly alters the active site structure but preserves 30% activity. In contrast, mutation to Glu otherwise preserves the active site structure but inactivates the enzyme. Thus, enzyme function correlates not with atom positions but with residue identity (chemistry), in this case. We observe strong destabilization of the Q69E-FeSOD oxidized state relative to the reduced state and intermediate destabilization of oxidized Q69H-FeSOD. Indeed, redox titrations indicate that mutation of Gln69 to His increases the reduction potential by 240 mV, whereas mutation to Glu appears to increase it by more than 660 mV. We find that this suffices to explain the mutants' loss of activity, although additional factors may also contribute. The strongly elevated reduction potential of Q69E-FeSOD may reflect reorganization of the active site H-bonding network, including possible reversal of the polarity of the key H-bond between residue 69 and coordinated solvent.
“…23–25 The coordinated solvent molecule is central to an active site H-bond network that connects it to bulk solvent via a second-sphere glutamine (Gln146 in E. coli MnSOD or Gln69 in E. coli FeSOD) which H-bonds with the hydroxyl of conserved Tyr34 which in turn H-bonds with a solvent molecule in the channel connecting the active site to bulk solvent (Figure 1). 15, 26–30 The most highly-conserved difference between FeSODs and MnSODs is the origin of the Gln residue (or in some cases His) 1, 3 that H-bonds to coordinated solvent. 5, 31–33 MnSODs contribute the conserved Gln146 from a position between a beta strands in the C-terminal domain (Figure 1) whereas FeSODs contribute Gln69 from an alpha helix in the N-terminal domain (Supplemental Figure S1).…”
The catalytic active site of Mn-specific SOD (MnSOD) is organized around a redox-active Mn ion. The most highly-conserved difference between MnSODs and the homologous FeSODs is the origin of a Gln in the second coordination sphere. In MnSODs it derives from the C-terminal domain whereas in FeSODs it derives from the N-terminal domain, yet its side chain occupies almost superimposable positions in the two types of SODs’ active sites. Mutation of this Gln69 to Glu in E. coli FeSOD increased the Fe3+/2+ reduction midpoint potential by > 0.6 V without disrupting the structure or Fe binding [E. Yikilmaz, D. W. Rodgers and A.-F. Miller (2006) Biochemistry 45(4) 1151–1161]. We now describe the analogous Q146E mutant of MnSOD, explaining its low Mn content in terms increased stability of the apo-Mn protein. In 0.8 M guanidinium HCl, the Q146E-apoMnSOD displays an apparent melting midpoint temperature (Tm) 35 °C higher that of WT-apoMnSOD, whereas the Tm of WT-holoMnSOD is only 20 °C higher than that of WT-apoMnSOD. In contrast, the Tm attributed to Q146E-holoMnSOD is 40 °C lower than that of Q146E-apoMnSOD. Thus our data refute the notion that the WT residues optimize structural stability of the protein, being instead consistent with conservation on the basis of enzyme function and therefore ability to bind metal ion. We propose that the WT-MnSOD protein conserves a destabilizing amino acid at position 146 as part of a strategy for favoring metal ion binding.
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