Roles of glutamates and metal ions in a rationally designed nitric oxide reductase based on myoglobin.
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 approximately 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 approximately 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 well-defined system to address important chemical and biological issues.
Lin
Ying-Wu
YW
Department of Chemistry, George L. Clark X-Ray Facility and 3M Materials Laboratory, and Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Yeung
Natasha
N
Gao
Yi-Gui
YG
Miner
Kyle D
KD
Tian
Shiliang
S
Robinson
Howard
H
Lu
Yi
Y
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D005971:Glutamates / Q000378:metabolism*; D007477:Ions; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D015202:Protein Engineering; D050661:Sperm Whale; D013997:Time Factors
D005971:Glutamates; D007477:Ions; D008670:Metals; D009211:Myoglobin; D009569:Nitric Oxide; D007501:Iron; D010088:Oxidoreductases; C034747:nitric-oxide reductase