This award in the Inorganic, Bioinorganic and Organometallic Chemistry program supports research by Professor Michael Maroney at the University of Massachusetts to understand the role of the protein in creating the active site in recombinant Streptomyces coelicolor nickel superoxide dismutase (NiSOD). Point mutations will be employed to address the following questions: 1. Do residues in the second coordination sphere of Ni play a critical role in optimizing the redox potential of the active site? 2. Is the quaternary structure required for catalysis? 3. What are the roles of conserved tyrosine residues near the active site? 4. How does the protein structure moderate the interaction of the active site with hydrogen peroxide, a molecule that is a reaction product, an active site reductant, and readily oxidizes thiolates, such as the cysteine ligands found in the active site? Mutant enzymes will be characterized using a strategy that employs a variety of complementary biophysical techniques including EPR and x-ray absorption spectroscopy. The effect of mutations on catalysis will be addressed by measuring the catalytic rate constant and examining the reaction mechanism using pulse radiolytic generation of superoxide and UV-Vis spectroscopy, and by assessing the redox potential of the enzyme using redox titrations. Mutations that result in a perturbed mechanism will be further examined using crystallography. Catalytic intermediates that have been detected in a number of existing mutant NiSODs will be freeze-trapped and examined spectroscopically
The study of NiSOD will contribute to the understanding of redox catalysis by biological nickel sites. The research will provide multidisciplinary training at the interface of chemistry and biology for students at all levels (undergraduate through post-doctoral).
Superoxide ion is a free radical that can generate reactive oxygen species, such as hydroxyl radical, in the body. These reactive oxygen species damage biological molecules and have been linked to aging, cancer, and neurodegenerative diseases. Superoxide dismutases are a diverse group of metalloenzymes that catalyze the conversion of superoxide ion to molecular oxygen and hydrogen peroxide at rates approaching the diffusion limit. As such, they constitute a cell's first line of defense against oxidative damage. Prior to 1999, three variations of superoxide dismutase had been extensively characterized. These enzymes contained copper (CuZnSOD), Manganese (MnSOD) or iron (FeSOD) as metals whose redox properties could be used in the reaction that is catalyzed. All of these metals will, in fact, catalyze the disproportionation of superoxide in water without the protein. In 1999, a nickel-dependent version of the enzyme was discovered in bacteria. This enzyme is unique in that nickel does not have the appropriate redox characteristics and does not catalyze the reaction in water without the protein. Thus, NiSOD represents a new design for this catalyst where the protein must confer the appropriate redox properties. Structural studies supported by NSF funding showed that the protein provides a unique active site composed of three N-terminal amino acids, His1 (provides an N-terminal amine ligation and ligation of the imidazole side chain), Cys2 (provides a backbone amidate and thiolate side chain as ligands), and Cys6, which provides a thiolate ligand (picture). The use of thiolate ligation was unprecedented among known SOD enzymes. Thus, NiSOD represents a case of convergent molecular evolution. The overall goals of the research conducted under this grant are to understand the completely different solution to how to control superoxide that is represented by NiSOD. This grant provided funding to address the roles of four of the nickel ligands in the enzyme, as well as a number of key amino acid residues in the second coordination sphere of the metal. The approach combined mutations of the recombinant enzyme and characterization of the mutants for reactivity by kinetic methods involving pulse radiolytic generation of superoxide, and for structure by crystallography, and various spectroscopic methods including magnetic circular dichroism and x-ray absorption. These studies showed that the Cys thiolate ligands are required for redox activity and are important to dictating the appropriate electronic structure for the nickel center. The role of the His1 N-terminal amine was addressed by an insertion mutant that placed an additional amino acid (Ala) at the N-terminus and thus moved the N-terminal amine. This mutation showed that the N-terminus was essential for generating the propoer structure of the reduced (NI(II)) form of the enzyme, which is essential for the redox process involved. Similarly, the mutation of the His1 residue to Ala not only revealed the role of Histidine in the redox process (stabilization of the oxidized, Ni(III), center, but also the importance of this ligand for the proper construction of the active site. They also revealed potential catalytic intermediates that are currently under investigation. These studies were extended to show the role of Glu17 in stabilizing the structure via H-bonding interactions with the imidazole side chain of His1. Another important result was the characterization of two mutants involving amino acid residues in the second coordination sphere by crystallography. The first, Y9F, revealed the anion (superoxide) binding site as a complex with bromide, and shows it to be removed from the nickel center (picture). The second, D3A, revealed a possible mechanism for redox cooperation between nickel centers in hexameric enzyme. Investigation of this mechanism is also a topic of current investigation. The last ligand to the nickel center is the backbone amidate from Cys2. This cannot be addressed by mutation, but we are pursuing a semi-synthetic strategy to address this last remaining ligand. The completion of this work will generate a rather complete picture of the mechanism employed by, and the design of, the NiSOD active site. The project contributes to the education and professional development of future scientists at all levels (undergraduate, graduate and post-graduate) and provides an opportunity to experience interdisciplinary science at the interface of chemistry and biology. The project also participates in an NSF-REU program that involved a student from an under-represented minority, and has benefited from the work of a faculty member from a four-year campus in the summer.
