Effect of outer-sphere side chain substitutions on the fate of the trans iron-nitrosyl dimer in heme/nonheme engineered myoglobins (FeBMbs): Insights into the mechanism of denitrifying NO reductases

• Published in: Biochemistry
• Main Authors: Matsumura, Hirotoshi, Chakraborty, Saumen, Reed, Julian, Lu, Yi, Moënne-Loccoz, Pierre
• Format: Text
• Language: English
• Published: 2016
• Subjects: Article; Sperm whale
• Online Access: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5181652/; http://www.ncbi.nlm.nih.gov/pubmed/27003474; https://doi.org/10.1021/acs.biochem.5b01109

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. FeBMb proteins, with their nonheme iron sites engineered into the heme distal pocket of sperm whale myoglobin, are attractive models to study the molecular details of the NO reduction reaction. Spectroscopic and structural studies of FeBMb 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 FeBMb 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 to a glutamic acid (i.e., I107E) decreases the formation rate of the five-coordinate heme-nitrosyl complex and allows for the sub-stoichiometric production of N2O. Here, we aim to better characterize the formation of the five-coordinate heme-nitrosyl complex and to explain why the N2O production increases with the I107E substitution. We follow the formation of the five-coordinate heme-nitrosyl inhibitory complex through the sequential exposure of FeBMb 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 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 FeBMb construct with an I107F substitution (FeBMb3) show an increased rate for the formation of the 5cLS heme-nitrosyl complex without N2O production. Taken together, our results suggest that production of N2O from the [6cLS heme ...