Introducing a 2-His-1-Glu Non-heme Iron Center into Myoglobin Confers Nitric Oxide Reductase Activity

摘要

A conserved 2-His-1-Glu metal center, as found in natural non-heme iron-containing enzymes, was engineered into sperm whale myoglobin by replacing Leu29 and Phe43 with Glu and His, respectively (swMb L29E, F43H, H64, called FeBMb(-His)). A high resolution (1.65 Å) crystal structure of Cu(II)-CN⁻ -FeBMb(-His) was determined, demonstrating that the unique 2-His-1-Glu metal center was successfully created within swMb. The FeBMb(-His) can bind Cu, Fe or Zn ions, with both Cu(I)-FeBMb(-His) and Fe(II)-FeBMb(-His) exhibiting nitric oxide reductase (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 N2O catalyzed by these two enzymes resulted in different intermediates; a five-coordinate heme-NO species was observed for Cu(I)-FeBMb(-His) due to the cleavage of the proximal heme Fe-His bond, while Fe(II)-FeBMb(-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 NOR family, gNOR.