Introducing a 2-His-1-Glu nonheme iron center into myoglobin confers nitric oxide reductase activity

A conserved 2-His-1-Glu metal center, as found in natural nonheme iron-containing enzymes, was engineered into sperm whale myoglobin by replacing Leu29 and Phe43 with Glu and His, respectively (swMb L29E, F43H, H64, called Fe _BMb-(-His)). A high resolution (1.65 Å) crystal structure of Cu(II)-CN ^--Fe _BMb(-His) was determined, demonstrating that the unique 2-His-1-Glu metal center was successfully created within swMb. The Fe _BMb(-His) can bind Cu, Fe, or Zn ions, with both Cu(I)-Fe _BMb(-His) and Fe(II)-Fe _BMb(-His) exhibiting nitric oxide reduc-tase (NOR) activities. Cu dependent NOR activity was significantly higher than that of Fe in the same metal binding site. EPR studies showed that the reduction of NO to N ₂O catalyzed by these two enzymes resulted in different intermediates; a five-coordinate heme-NO species was observed for Cu(I)-Fe _BMb(-His) due to the cleavage of the proximal heme Fe-His bond, while Fe(II)-Fe _BMb-(-His) remained six-coordinate. Therefore, both the metal ligand, Glu29, and the metal itself, Cu or Fe, play crucial roles in NOR activity. This study presents a novel protein model of NOR and provides insights into a newly discovered member of the NOR family, gNOR.