Rational design of a structural and functional nitric oxide reductase

Abstract

SummaryProtein design provides an ultimate test of our knowledge about proteins and allows the creation of novel enzymes for biotechnological applications. While progress has been made in designing proteins that mimic native proteins structurally1–3, it is more difficult to design functional proteins4–8. In comparison to recent successes in designing non-metalloproteins4,6,7,9,10, it is even more challenging to rationally design metalloproteins that reproduce both the structure and function of native metalloenzymes5,8,11–20, since protein metal binding sites are much more varied than non-metal containing sites, in terms of different metal ion oxidation states, preferred geometry and metal ion ligand donor sets. Because of their variability, it has been difficult to predict metal binding site properties in silico, as many of the parameters for metal binding sites, such as force fields are ill-defined. Therefore, the successful design of a structural and functional metalloprotein will greatly advance the field of protein design and our understanding of enzymes. Here, we report a successful, rational design of a structural and functional model of a metalloprotein, nitric oxide reductase (NOR), by introducing three histidines and one glutamate, predicted as ligands in the active site of NOR, into the distal pocket of myoglobin. A crystal structure of the designed protein confirms that the minimized computer model contains a heme/non-heme FeB center that is remarkably similar to that in the crystal structure. This designed protein also exhibits NOR activity. This is the first designed protein that models both the structure and function of NOR, offering insight that the active site glutamate is required for both iron binding and activity. These results show that structural and functional metalloproteins can be rationally designed in silico.