Significance. We aim to determine the essential structural and thermodynamic features which govern enzymatic nitric oxide detoxification. We use a cycle of computational design and biochemical analysis of an artificial nitric oxide dioxygenase (NOD) formed by combining an artificial heme-based oxygen binding protein domain with a flavoprotein reductase domain derived from nature. Use of such a robust, simple protein makes it significantly easier to make both small- and large-scale changes to the protein and positively identify critical features necessary for enzyme function. Nitric oxide plays a central role in many signaling process in human biology, yet due to its degree of chemical reactivity it has also been implicated in a surprising number of serious disorders such as Lou Gehrig's Disease and ischemic brain injury. A superior nitric oxide dioxygenase thus promises to be useful in future treatments of many pathological conditions. Conversely, unwanted NOD activity has produced severe complications in hemoglobin-based blood substitutes, and it is important to learn how to reduce or eliminate NOD activity in these therapeutics without adversely affecting oxygen binding. Innovation. This catalytic construct represents the next generation in protein design, moving design technology from the current focus on simple protein domains with single cofactors to significantly more challenging and sophisticated multidomain structures that more closely resemble the complex assemblies seen in nature. This project has the capacity to dramatically advance two important technologies: hemoglobin-based blood substitutes and enzyme therapeutics. First, lessons learned in this project promise to revitalize the field of hemoglobin-based blood substitutes, enabling both the reengineering of native hemoglobins and the creation of entirely new oxygen transport proteins minimally reactive with nitric oxide while still carrying oxygen. Second, a synthetic enzyme has the potential to transform the field of enzyme therapy because of the many advantages designed enzymes have over their natural counterparts, most importantly the ability to utilize non-natural cofactors better optimized for the target activity and their greatly increased stability over natural protein (53). This project thus represents a new direction in enzyme therapy, and our design pathway is an enabling technology which will be used by us and others in the creation of future enzyme therapeutics. Specific Aims. This work will allow us to answer some important questions about this enzyme: Aim 1. What role does the heme reduction potential play in the nitric oxide dioxygenase reaction? Aim 2. How important are electron transfer dynamics and thermodynamics in this reaction? Aim 3. How do protein dynamics and structure govern NOD function?