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标题：Introduction and characterization of a functionally linked metal ion binding site at the exposed heme edge of myoglobin
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作者：Christie L. Hunter ; Robert Maurus ; Marcia R. Mauk 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2003
卷号：100
期号：7
页码：3647-3652
DOI：10.1073/pnas.0636702100
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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 1H NMR spectroscopy indicates that Mn2+ binds in the vicinity of the heme 6-propionate as anticipated, and potentiometric titrations establish that the affinity of the new site for Mn2+ is 1.28(4) x 104 M-1 (pH 6.96, ionic strength I = 17.2 {micro}M, 25{degrees}C). In addition, these substitutions lower the reduction potential of the protein and increase the pKa 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 Mn2+-peroxidase activity of the variant are both increased {approx}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 Mn2+. The structure of the variant has been determined by x-ray crystallography to define the coordination environment of bound Mn2+ and Cd2+. 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.