title: Effect of Outer-Sphere Side Chain Substitutions on the Fate of the trans Iron-Nitrosyl Dimer in Heme/Nonheme Engineered Myoglobins (Fe(B)Mbs): Insights into the Mechanism of Denitrifying NO Reductases.
abstract: Denitrifying NO reductases are transmembrane protein complexes that utilize a heme/nonheme diiron center at their active sites to reduce two NO molecules to the innocuous gas N2O. Fe(B)Mb proteins, with their nonheme iron sites engineered into the heme distal pocket of sperm whale myoglobin, are attractive models for studying the molecular details of the NO reduction reaction. Spectroscopic and structural studies of Fe(B)Mb constructs have confirmed that they reproduce the metal coordination spheres observed at the active site of the cytochrome c-dependent NO reductase from Pseudomonas aeruginosa. Exposure of Fe(B)Mb to excess NO, as examined by analytical and spectroscopic techniques, results primarily in the formation of a five-coordinate heme-nitrosyl complex without N2O production. However, substitution of the outer-sphere residue Ile107 with a glutamic acid (i.e., I107E) decreases the formation rate of the five-coordinate heme-nitrosyl complex and allows for the substoichiometric production of N2O. Here, we aim to better characterize the formation of the five-coordinate heme-nitrosyl complex and to explain why the level of N2O production increases with the I107E substitution. We follow the formation of the five-coordinate heme-nitrosyl inhibitory complex through the sequential exposure of Fe(B)Mb to different NO isotopomers using rapid-freeze-quench resonance Raman spectroscopy. The data show that the complex is formed by the displacement of the proximal histidine by a new NO molecule after the weakening of the Fe(II)-His bond in the intermediate six-coordinate low-spin (6cLS) heme-nitrosyl complex. These results lead us to explore diatomic migration within the scaffold of myoglobin and whether substitutions at residue 107 can be sufficient to control access to the proximal heme cavities. Results on a new Fe(B)Mb construct with an I107F substitution (Fe(B)Mb3) show an increased rate for the formation of the five-coordinate low-spin heme-nitrosyl complex without N2O production. Taken together, our results suggest that production of N2O from the [6cLS heme {FeNO}(7)/{Fe(B)NO}(7)] trans iron-nitrosyl dimer intermediate requires a proton transfer event facilitated by an outer-sphere residue such as E107 in Fe(B)Mb2 and E280 in P. aeruginosa cNOR.
authors.affiliation: Division of Environmental and Biomolecular Systems, Institute of Environmental Health, Oregon Health & Science University , Portland, Oregon 97239, United States.
authors.forename: Hirotoshi
authors.initials: H
authors.lastname: Matsumura
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Saumen
authors.initials: S
authors.lastname: Chakraborty
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Julian
authors.initials: J
authors.lastname: Reed
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.|Department of Biochemistry, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Yi
authors.initials: Y
authors.lastname: Lu
authors.affiliation: Division of Environmental and Biomolecular Systems, Institute of Environmental Health, Oregon Health & Science University , Portland, Oregon 97239, United States.
authors.forename: Pierre
authors.initials: P
authors.lastname: Moënne-Loccoz
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D020134:Catalytic Domain; D019281:Dimerization; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D009154:Mutation; D009211:Myoglobin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019159:Nonheme Iron Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering; D021122:Protein Subunits; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013059:Spectrum Analysis, Raman; D050661:Sperm Whale
chemical_list: D001426:Bacterial Proteins; D009211:Myoglobin; D019159:Nonheme Iron Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D006418:Heme; D010088:Oxidoreductases; C034747:nitric-oxide reductase