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

Abstract

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.

Assessment of crude oil degradation efficiency of newly isolated actinobacteria reveals untapped bioremediation potentials

“…The biodegradation broths were centrifuged at 10,000 rpm for 20 min at 4 C. The clear supernatants were recovered and assayed for total peroxidase activity according to the method of Hunter et al (2003). Peroxidase activity was determined via oxidation of 0.24 mM 2,2 0 azino-di-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) buffered with 50 mM sodium acetate buffer pH 5 in the presence of 5 mM H 2 O 2 at 414 nm for 5 min on a ultraviolet/visible (UV/Vis) spectrophotometer.…”

New conjugates of superoxide dismutase/catalase mimetics with cyclodestrins

“…In order to quantify the peroxidase activity of salen cyclodextrin conjugates, ABTS was used as the substrate [56][57][58]. The reaction of ABTS with H 2 O 2 in the presence of 4a or 4b generates ABTS Å+ :…”

Contribution of the heme propionate groups to the electron transfer and electrostatic properties of myoglobin

“…Mb is readily autoxidized to metMb in an aerobic environment (reviewed in [9]), and enzymatic reduction of myoglobin to the reduced, Fe(II) state was reported over twenty years ago [10] though the subsequent literature has produced inconsistent results regarding the nature of this enzymatic system (reviewed in [11]). The literature concerning the oxidation-reduction properties and electron transfer kinetics of myoglobin is extensive (a review of the older literature is provided in [12]) and in more recent years has emphasized the use of myoglobin as (i) a model for studies of intramolecular electron transfer (e.g., [13,14]), (ii) a participant in protein-protein electron transfer reactions (e.g., [15][16][17][18]), a model for ligand binding (e.g., [19][20][21][22][23]) and (iv) a protein scaffold for genetic engineering of new functionalities (e.g., [19,[24][25][26][27][28]). …”