Deciphering the Catalytic Mechanism of Human Manganese Superoxide Dismutase

The livelihood of human cells is heavily dependent on the ability to modulate the presence of highly reactive oxygen-based molecules termed reactive oxygen species (ROS). In excess, ROS facilitate oxidative damage to the macromolecules of cellular life. SODs are the major family of antioxidant proteins that prevent the buildup of overwhelming amounts of ROS within cells. Sometimes dubbed the “first line of defense” against oxidative damage, SODs defend against the harmful accumulation of ROS by eliminating superoxide. Superoxide is a ROS itself that is also a precursor to much more harmful ROS molecules. MnSOD is the manganese containing form of human SODs that dwells within the mitochondria and is responsible for protecting the organelle against superoxide-mediated damage. The protein is arguably the most significant antioxidant enzyme as the mitochondria are especially integral for cellular vitality. This is exemplified by the embryonic lethality of mice lacking MnSOD and the multitude of human disease states that manifest as a result of dysfunctional MnSOD. The bioprotective attributes of MnSOD have attracted the attention of clinicians and is illustrated by the multiple ongoing clinical trials that attempt to mimic the function of the enzyme. While MnSOD has proven to be of significant importance for human vitality and has been studied extensively since its discovery over 50 years ago, its atom-by-atom mechanism has still been elusive and the mechanism of MnSOD has yet to be defined due to its nature of catalysis. MnSOD performs its function through concerted proton-electron transfers (CPETs) at specific sites of the enzyme that have been extremely difficult to detect experimentally. An emerging biophysical tool capable of circumventing previous experimental obstacles is neutron protein crystallography. This method involves diffracting neutrons off of crystallized protein samples with controlled electronic states into a pattern that can be deciphered for specific proton sites thereby permitting the experimental coupling of proton and electron transfers. In this thesis work, significant revelations are made for the mechanism of MnSOD using a multitude of approaches, including neutron crystallography where significant developments are also made for the emerging technique

Filamentation Involves Two Overlapping, but Distinct, Programs of Filamentation in the Pathogenic Fungus Candida albicans

The ability of the human pathogenic fungus Candida albicans to switch between yeast-like and filamentous forms of growth has long been linked to pathogenesis. Numerous environmental conditions, including growth at high temperatures, nutrient limitation, and exposure to serum, can trigger this morphological switch and are frequently used in in vitro models to identify genes with roles in filamentation. Previous work has suggested that differences exist between the various in vitro models both in the genetic requirements for filamentation and transcriptional responses to distinct filamentation-inducing media, but these differences had not been analyzed in detail. We compared 10 in vitro models for filamentation and found broad genetic and transcriptomic differences between model systems. The comparative analysis enabled the discovery of novel media-independent genetic requirements for filamentation as well as a core filamentation transcriptional profile. Our data also suggest that the physical environment drives distinct programs of filamentation in C. albicans, which has significant implications for filamentation in vivo

A Review of the Catalytic Mechanism of Human Manganese Superoxide Dismutase

Superoxide dismutases (SODs) are necessary antioxidant enzymes that protect cells from reactive oxygen species (ROS). Decreased levels of SODs or mutations that affect their catalytic activity have serious phenotypic consequences. SODs perform their bio-protective role by converting superoxide into oxygen and hydrogen peroxide by cyclic oxidation and reduction reactions with the active site metal. Mutations of SODs can cause cancer of the lung, colon, and lymphatic system, as well as neurodegenerative diseases such as Parkinson’s disease and amyotrophic lateral sclerosis. While SODs have proven to be of significant biological importance since their discovery in 1968, the mechanistic nature of their catalytic function remains elusive. Extensive investigations with a multitude of approaches have tried to unveil the catalytic workings of SODs, but experimental limitations have impeded direct observations of the mechanism. Here, we focus on human MnSOD, the most significant enzyme in protecting against ROS in the human body. Human MnSOD resides in the mitochondrial matrix, the location of up to 90% of cellular ROS generation. We review the current knowledge of the MnSOD enzymatic mechanism and ongoing studies into solving the remaining mysteries

Perfect Crystals: microgravity capillary counterdiffusion crystallization of human manganese superoxide dismutase for neutron crystallography

Abstract The NASA mission Perfect Crystals used the microgravity environment on the International Space Station (ISS) to grow crystals of human manganese superoxide dismutase (MnSOD)—an oxidoreductase critical for mitochondrial vitality and human health. The mission’s overarching aim is to perform neutron protein crystallography (NPC) on MnSOD to directly visualize proton positions and derive a chemical understanding of the concerted proton electron transfers performed by the enzyme. Large crystals that are perfect enough to diffract neutrons to sufficient resolution are essential for NPC. This combination, large and perfect, is hard to achieve on Earth due to gravity-induced convective mixing. Capillary counterdiffusion methods were developed that provided a gradient of conditions for crystal growth along with a built-in time delay that prevented premature crystallization before stowage on the ISS. Here, we report a highly successful and versatile crystallization system to grow a plethora of crystals for high-resolution NPC