Structural insights into a low-spin myoglobin variant with bis-histidine coordination from molecular modeling

Rational design of functional enzymes is a powerful strategy to gain deep insights into more complex native enzymes, such as nitric oxide reductase (NOR). Recently, we engineered a functional model of NOR by creating a two His and one Glu (2-His-1-Glu) non-heme iron center in sperm whale myoglobin (swMb L29E, F43H, H64, called Fe(B)Mb(-His)). It was found that Fe(B) Mb(-His) adopts a low-spin state with bis-His coordination in the absence of metal ions binding to the designed metal center. However, no structural information was available for the variant in this special spin state. We herein performed molecular modeling of Fe(B)Mb(-His) and compared with the X-ray structure of its copper bound derivative, Cu(II)-CN(-)-Fe(B) Mb(-His), resolved recently at a high resolution (1.65 Å) (PDB entry 3MN0). The simulated structure shows that mutation of Leu to Glu at position 29 in the hydrophobic heme pocket alters the folding behavior of Mb. The hydrogen bond between Glu29 and His64 further plays a role in stabilizing the bis-His (His64/His93) coordination structure. This study offers an excellent example of using molecular modeling to gain insights in rational design of both structural and functional proteins.