Role of Conserved Tyrosine Residues in NiSOD Catalysis: A Case of Convergent Evolution
2009
Superoxide dismutases (SODs) are enzymes that catalyze the conversion of superoxide to molecular oxygen and hydrogen peroxide (Equation 1–3), and are thus part of cellular defenses against damage due to reactive oxygen species (ROS)(1–4).
M(n+1)+O2•-→M(n)+O2
(1)
M(n)+2H++O2•-→M(n+1)+H2O2
(2)
2O2•-+2H+→O2+H2O2
(3)
SODs utilize one-electron redox-active metal centers to carry out catalysis. There are four known SODs categorized by the metal present: (1) Cu/Zn, (2) Mn, (3) Fe and (4) Ni. These four SODs fall into three protein classes based on amino acid sequence homology, with Fe and Mn in the same class. In addition to having no sequence homology with other SODs(5), NiSOD has other notable differences: Nickel is the only metal found in a SOD where the aquated ion does not react with O2−(1), presumably because the redox potential of Ni(OH2)62+/3+ (> 2 V)(6) lies outside of the potentials for oxidation and reduction of O2−. In SODs, the protein component serves at least three important functions with regard to catalysis: to adjust the redox potential of the metallocenter(7–10), to provide a source of protons(11, 12), and to control the access of anions to the active site(13–16). In NiSOD, the mechanism by which the protein tunes the redox potential of nickel is also distinct from other SODs in that it is the only metal center that employs cysteine thiolate ligands(17–21), which at first glance would seem to be incompatible with a catalytic center that produces oxygen and peroxide(22–24).
Crystal structures of NiSOD reveal a five-coordinate pyramidal site that is associated with an oxidized Ni(III) center (Figure 1), and a four-coordinate planar site associated with a reduced Ni(II) center(17, 21). The nickel ligands derive from three amino acid residues: the N-terminal amino group of His1, an amidate N-donor from Cys2 and sidechain thiolates from Cys2 and Cys6. The fifth ligand in the Ni(III) site is provided by the imidazole sidechain of His1 (His-on), which is unbound in the Ni(II) (His-off) site(17, 21).
Figure 1
Active site from one monomer of the homohexameric NiSOD from Streptomyces coelicolor, showing the first nine residues and Tyr62(17). The nickel is shown in the five-coordinate pyramidal oxidized His-on form. PDB code 1T6U. Image was generated in PyMOL ...
There are four tyrosine residues in the amino acid sequence of Streptomyces coelicolor NiSOD, two of which (Tyr9 and Tyr62) are conserved in nearly all NiSODs (see supporting information). Tyr9 is positioned near the vacant coordination site opposite the His1 imidazole ligand in the His-on structure, (O-Ni = 5.47 A) and is involved in H-bonding with two ordered water molecules in the active site, neither of which is a nickel ligand (O-Ni = 2.57, 3.15 A). These water molecules are also H-bonded to amide protons from Cys6 and Asp3. The other conserved tyrosine, Tyr62, is near Tyr9 and ~13 A away from the nickel center.
The SOD proteins supply protons for the production of H2O2 and control anion access to the active site(7–10). NiSOD lacks an aqua ligand that has been associated with redox tuning and proton supply(8) in MnSOD and FeSOD, but the thiolate ligands may serve an analogous role(25, 26). Anion access is controlled in SODs using a combination of size constraint(27), electrostatic steering(28–30) and, in the cases of MnSOD(12, 31, 32), and FeSOD(15, 33) a “gatekeeper” tyrosine and neighboring phenylalanine(27). Herein, we probe the role of the conserved tyrosine residues and the aspartate (Asp3) in the Ni hook domain, for insights into the NiSOD mechanism using a combination of mutagenesis to systematically perturb the tyrosine residues, crystallographic and spectroscopic studies to assess the structural ramifications of the mutations, and kinetics using pulse-radiolytic generation of superoxide to examine function. The mutation of Tyr9 led to the serendipitous isolation and the first crystallographic characterization of an anion complex of NiSOD (Cl− and Br−). In addition to intersubunit interactions, Asp3 also affects the position of Tyr9 in a way that changes the function of the active site. The results point to a role for Tyr9 in the mechanism for regulating anion access that has many features in common with other SODs, particularly MnSOD. Thus, NiSOD represents a fascinating case of molecular convergent evolution.
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