Large Volume Crystal Growth of Superoxide Dismutase Complexes in Microgravity for Neutron Diffraction Studies

Superoxide dismutases (SODs) are important antioxidant enzymes that protect all living cells against toxic oxygen metabolites, also known as Reactive Oxygen Species (ROS). SODs are one of the fastest known enzymes and are rate-limited only by the diffusion of the substrate and products. SODs are the first line of defense to protect organisms against metabolic- and ionizing radiation-induced ROS. SOD protects cells by dismuting two molecules of superoxide anions to form hydrogen peroxide and molecular oxygen via a cyclic oxidation-reduction reaction. SODs contain metal ions in their active sites. Humans have Cu/ZnSOD in the cytosol and extracellular spaces and MnSOD in their mitochondria. Mutations in SOD lead to degenerative diseases such as amyotrophic lateral sclerosis (ALS), diabetes, and cancer. This proposal will study SODs from the model system Escherichia coli as they are easy to produce, stable, and the active sites are identical with human homologs. Bacteria have both Fe and MnSOD. Despite the biological and medical importance of SOD, the enzymatic mechanism is still unknown. Precise structural data are needed to understand the enzymatic mechanism of SOD. The binding sites of the diatomic substrate and product as well as the source of the protons in the reaction have been studied but their exact identification has not been possible. This detailed information can only be determined by neutron diffraction. Complexes of the Fe and MnSOD including structural intermediates and mutants will be the targets for large volume crystal (≥ 1 mm cubed) growth for structure determination by Neutron Macromolecular Crystallography (NMC). The quiescent environment afforded by microgravity is known to grow crystals large enough for neutron studies and in 2001, the Borgstahl laboratory successfully grew large crystals of SOD using microgravity conditions on the International Space Station (ISS). With NASA’s renewed interest in implementing the microgravity environment on the ISS for protein crystal growth we would like to move forward with these exciting early microgravity crystallization results for SOD. Existing crystallization facilities, such as the Granada Box Facility (GBF) that employs capillary counterdiffusion protocols or the Protein Crystallization Facility (PCF) that uses vapor diffusion methods will be used to achieve these goals. A microgravity environment is essential to form a stable supersaturation gradient to obtain the large crystals required for NMC. NMC will be performed with collaborators at Oak Ridge National Laboratory. The principal outcome will be to identify the role of hydrogen atoms in enzymatic activity, discern superoxide from peroxide and water from hydroxide ion by their protonation state and decipher a structure-based mechanism for Mn and FeSODs more precisely than from previous X-ray crystallographic models determined from Earth-grown crystals. These contributions will provide criteria needed for protein engineering desirable properties into enzymatic metal centers.

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