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.

Roles of glutamates and metal ions in a rationally designed nitric oxide reductase based on myoglobin

“…To probe the conformational changes of introducing the second Glu (E107), we performed a structural alignment of Fe (II)-I107E Fe B Mb and the previously reported FeðIIÞ-Fe B Mb (14). The comparison, shown in Fig.…”

“…Important issues such as the roles of the conserved glutamates and nonheme Fe B in NOR have been previously addressed using biochemical and biophysical studies or biomimetic modeling (24, 25, 27-37, 45, 56, 57). As a complementary approach, rational protein design, using small, easy-toproduce and well-characterized proteins such as myoglobin, offers a powerful method with which to gain insights into more complex native enzymes such as NOR (14). Similar to synthetic models (41,43), the metal ion at the putative Fe B site in the protein model can be substituted freely.…”

“…Although designed proteins that model the structure of native enzymes have been known for a while (4)(5)(6)(7)(8)(9)(10), successful designs of proteins that mimic both the structure and function of native enzymes have been reported only recently (11)(12)(13)(14)(15)(16). While being able to design such functional proteins is laudable, the impact of such an achievement would be greater if the designed proteins can be used to address fundamental issues in chemistry and biology that are difficult to tackle by other methods.…”

“…We have recently designed a structural and functional protein model of bacterial NOR by engineering three histidines and one glutamate into the distal pocket of sperm whale myoglobin (swMb, L29H, F43H, H64, and V68E, named Fe B Mb) (14). Like synthetic models, this "bottom-up" approach complements the "top-down" approach of the study of native NOR in that it provides insights into whether certain "necessary" structural elements are enough to impart enzyme function.…”

“…More importantly, the rigid protein network allows precise placement of either glutamate or metal ions in myoglobin to address their roles in NOR activity. Toward this goal, we have demonstrated that both the histidines and one of the glutamates are essential for iron binding and NO reduction activity (14). However, the role of the second Glu close to the Fe B site and the role of different metal ions in the Fe B site have not been addressed.…”