Engineered metalloregulation of azide binding affinity and reduction potential of horse heart myoglobin

Abstract

Metal ion binding to a previously reported variant of horse heart myoglobin (Lys45Glu/Lys63Glu) with a metal ion binding site on the surface of the protein that is adjacent to the haem binding site has been shown to influence ligand binding and electrochemical properties of the protein. For example, the K(d) (μM) for binding of azide to this variant decreases from 277 ± 9 to 32 ± 3 following addition of a saturating concentration of Mn(2+) (the value for the wild-type protein under the same conditions is 26 ± 1). Similarly, the midpoint reduction potential E(m) (mV vs. standard hydrogen electrode) increases from 9 to 40 in the presence of a saturating concentration of Mn(2+) (the value for the wild-type protein under the same conditions is 45 ± 2). These results demonstrate the potential value of engineered metal ion binding sites as a means of regulating the functional properties of even simple haem proteins.

Change in structure and ligand binding properties of hyperstable cytochrome c555 from Aquifex aeolicus by domain swapping

“…The function of a protein is related to its three‐dimensional structure, and many attempts have been made to alter the structure, including mutagenesis and chemical modification . However, since the amino acid sequence defines the unique three‐dimensional structure of a protein, conversion of the function of a protein without modifying its amino acid sequence is limited.…”

“…The function of a protein is related to its threedimensional structure, and many attempts have been made to alter the structure, including mutagenesis and chemical modification. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17] However, since the amino acid sequence defines the unique threedimensional structure of a protein, conversion of the function of a protein without modifying its amino acid sequence is limited. An example of a native bifunctional heme protein is dehaloperoxidase, which has been shown to convert with its substrate radical from an O 2 -binding hemoglobin to a peroxidase.…”