Introduction and characterization of a functionally linked metal ion binding site at the exposed heme edge of myoglobin.
A binding site for metal ions has been created on the surface of horse heart myoglobin (Mb) near the heme 6-propionate group by replacing K45 and K63 with glutamyl residues. One-dimensional (1)H NMR spectroscopy indicates that Mn(2+) binds in the vicinity of the heme 6-propionate as anticipated, and potentiometric titrations establish that the affinity of the new site for Mn(2+) is 1.28(4) x 10(4) M(-1) (pH 6.96, ionic strength I = 17.2 microM, 25 degrees C). In addition, these substitutions lower the reduction potential of the protein and increase the pK(a) for the water molecule coordinated to the heme iron of metmyoglobin. The peroxidase [2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid), ABTS, as substrate] and the Mn(2+)-peroxidase activity of the variant are both increased approximately 3-fold. In contrast to wild-type Mb, both the affinity for azide and the midpoint potential of the variant are significantly influenced by the addition of Mn(2+). The structure of the variant has been determined by x-ray crystallography to define the coordination environment of bound Mn(2+) and Cd(2+). Although slight differences are observed between the geometry of the binding of the two metal ions, both are hexacoordinate, and neither involves coordination by E63.
Hunter
Christie L
CL
Department of Biochemistry and Molecular Biology and Protein Engineering Network of Centres of Excellence, University of British Columbia, Vancouver, BC, Canada V6T 1Z3.
Maurus
Robert
R
Mauk
Marcia R
MR
Lee
Hung
H
Raven
Emma L
EL
Tong
Harry
H
Nguyen
Nham
N
Smith
Michael
M
Brayer
Gary D
GD
Mauk
A Grant
AG
D000818:Animals; D001665:Binding Sites; D002712:Chlorides / Q000737:chemistry; D018360:Crystallography, X-Ray / Q000379:methods; D004563:Electrochemistry / Q000379:methods; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006736:Horses; D007700:Kinetics; D017895:Manganese Compounds / Q000737:chemistry; D008670:Metals / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis; D009211:Myoglobin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods
D002712:Chlorides; D017895:Manganese Compounds; D008670:Metals; D009211:Myoglobin; D011994:Recombinant Proteins; D006418:Heme; C025340:manganese chloride