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

Abstract

A structural and functional model of bacterial nitric oxide reductase (NOR) has been designed by introducing two glutamates (Glu) and three histidines (His) in sperm whale myoglobin. X-ray structural data indicate that the three His and one Glu (V68E) residues bind iron, mimicking the putative Fe B site in NOR, while the second Glu (I107E) interacts with a water molecule and forms a hydrogen bonding network in the designed protein. Unlike the first Glu (V68E), which lowered the heme reduction potential by ∼110 mV, the second Glu has little effect on the heme potential, suggesting that the negatively charged Glu has a different role in redox tuning. More importantly, introducing the second Glu resulted in a ∼100% increase in NOR activity, suggesting the importance of a hydrogen bonding network in facilitating proton delivery during NOR reactivity. In addition, EPR and X-ray structural studies indicate that the designed protein binds iron, copper, or zinc in the Fe B site, each with different effects on the structures and NOR activities, suggesting that both redox activity and an intermediate five-coordinate heme-NO species are important for high NOR activity. The designed protein offers an excellent model for NOR and demonstrates the power of using designed proteins as a simpler and more welldefined system to address important chemical and biological issues.biomimetic models | heme-copper oxidase | metalloprotein | protein design | protein engineering R ational design of proteins that mimic both structure and function of more complex native enzymes has been a long soughtafter goal, as the process is an ultimate test of our knowledge and an excellent means to develop advanced biocatalysts (1-3). Although designed proteins that model the structure of native enzymes have been known for a while (4-10), successful designs of proteins that mimic both the structure and function of native enzymes have been reported only recently (11-16). 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. One primary example is the roles of conserved glutamates and metal ions in bacterial nitric oxide reductase (NOR) (17)(18)(19).NO is critical for all life (20). Bacterial denitrification is a crucial part of the nitrogen cycle in nature that involves a four-step, five-electron reduction of nitrate (NO 3 − ) to dinitrogen (N 2 ) (17, 19). Bacterial NOR is a membrane-bound protein that catalyzes one step of this process, namely, the two-electron reduction of NO to N 2 O (17, 19). With no crystal or solution structure available for bacterial NOR to date, sequence alignments and homology modeling (21, 22) have indicated that NOR is structurally homologous to the largest subunit (subunit I) of hemecopper oxidases (HCOs) (23), enzymes that catalyze reduction of O 2 to water. The active sites of both NOR and HCO contain a proximal histidine-coo...

Dynamics comparison of two myoglobins with a distinct heme active site

“…During the last few decades, swMb has been well studied as a represent heme protein. In addition, it has been widely used as a protein model for rational design of artificial heme proteins with novel functions [1,4,[8][9][10][11]. Comparatively, the structureactivity relationship has not been fully understood for the unique apMb [6,[12][13][14][15][16].…”

Steady-state kinetics with nitric oxide reductase (NOR): New considerations on substrate inhibition profile and catalytic mechanism

“…Besides inorganic model compounds [32] (and references therein), an engineered sperm whale myoglobin was constructed [33,34]. It successfully reproduced the NOR active centre with the magnetic coupling between the two iron sites, but experiments carried out in the presence of NO have shown low reductase activity [33][34][35].…”

The Outer-Coordination Sphere: Incorporating Amino Acids and Peptides as Ligands for Homogeneous Catalysts to Mimic Enzyme Function

“…They also demonstrated that adding a second glutamic acid was critical. This Glu is seen in the native enzyme and increased reactivity by 100% in the artificial enzyme, which the authors ascribe to a hydrogen bond between the Glu and water-promoting proton transfer (199). Furthermore, they demonstrated that the metal at this site can both structurally and electrochemically tune the active site.…”