Formation of sulphmyoglobin during expression of horse heart myoglobin in <i>Escherichia coli</i>

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) ؋ 10 4 M ؊1 (pH 6.96, ionic strength I ‫؍‬ 17.2 M, 25°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 Ϸ3-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.

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“…The expression (16,17), purification (17), and mutagenesis (18) of horse heart Mb were performed as described.…”

Section: Methodsmentioning

confidence: 99%

Introduction and characterization of a functionally linked metal ion binding site at the exposed heme edge of myoglobin

Hunter

Maurus²,

Mauk³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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“…Expression of a synthetic gene coding for horse heart Mb and preparation of variants by site-directed mutagenesis has been described . The resulting protein was reconstituted with fresh protoheme IX (Porphyrin Products, Logan, UT) to remove any sulfMb formed during fermentation .…”

Section: Methodsmentioning

confidence: 99%

Efficient Coupled Oxidation of Heme by an Active Site Variant of Horse Heart Myoglobin

et al. 1996

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The coupled oxidation of protoheme IX at the active sites of heme proteins exposed to ascorbate in the presence of dioxygen has been known for over 65 years and is related to the catalytic conversion of protoheme IX to biliverdin by the enzyme heme oxygenase. The present report demonstrates that replacement of two residues present in the distal heme pocket of horse heart myoglobin (Mb), Val67 and Val68, with alanine and serine, respectively, increases the efficiency of coupled oxidation in the active site of myoglobin. HPLC analysis of the heme oxidation product demonstrates that the specificity of wild-type myoglobin for opening the heme ring at the R-meso carbon is retained by the variant. The infrared spectrum of the carbonyl derivative of the variant exhibits ν CO bands of comparable intensity at 1948 and 1958 cm -1 , which are consistent with the presence of the polar serine residue in close proximity to the bound ligand. The high-resolution crystal structure of the Val67Ala/Val68Ser metMb variant establishes that a hydrogen bond forms between the hydroxyl group of Ser68 and the coordinated water molecule. The increased rate of coupled oxidation exhibited by the variant is attributed to the increased polarity of the distal pocket created by the amino acid substitutions. These results are discussed in light of recent work concerning the active site of heme oxygenase.

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“…Recombinant wild-type horse heart myoglobin and the His-64 Thr variant were prepared as described previously [22][23][24].…”

Section: Experimental Mutagenesis and Protein Purificationmentioning

confidence: 99%

Structural and spectroscopic studies of azide complexes of horse heart myoglobin and the His-64→Thr variant

Maurus

Bogumil

Nguyen

et al. 1998

Biochemical Journal

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The high-resolution X-ray crystallographic structures of horse heart azidometmyoglobin complexes of the wild-type protein and the His-64-->Thr variant have been determined to 2.0 and 1.8 A respectively. Azide binds to wild-type metmyoglobin in a bent configuration with an Fe-N-1-N-3 angle of 119 degrees and is oriented into the distal crevice in the direction of Ile-107. The proximity of the His-64 NE2 atom to the N-1 atom of the bound azide indicates stabilization of the ligand by the His-64 side chain through hydrogen bonding. In addition, structural characterization of wild-type horse heart azidometmyoglobin establishes that the only structural change induced by ligand binding is a small movement of the Leu-29 side chain away from the azide ligand. EPR and Fourier transform infrared spectroscopy were used to characterize the myoglobin azide complexes further. EPR spectroscopy revealed that, in contrast with wild-type azidometmyoglobin, two slightly different low-spin species are formed by azide bound to the His-64-->Thr variant both in solution and in a polycrystalline sample. One of these low-spin species has a greater relative intensity, with g values very similar to those of the azide complex of the wild-type protein. These EPR results together with structural information on this variant indicate the presence of two distinct conformations of bound azide, with one form predominating. The major conformation is comparable to that formed by wild-type myoglobin in which azide is oriented into the distal crevice. In the minor conformation the azide is oriented towards the exterior of the protein.

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Formation of sulphmyoglobin during expression of horse heart myoglobin in Escherichia coli

Cited by 19 publications

References 26 publications

Introduction and characterization of a functionally linked metal ion binding site at the exposed heme edge of myoglobin

Introduction and characterization of a functionally linked metal ion binding site at the exposed heme edge of myoglobin

Efficient Coupled Oxidation of Heme by an Active Site Variant of Horse Heart Myoglobin

Structural and spectroscopic studies of azide complexes of horse heart myoglobin and the His-64→Thr variant

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