FTIR Analysis of the Interaction of Azide with Horse Heart Myoglobin Variants

Abstract

The interaction of azide with variants of horse heart myoglobin (Mb) has been characterized by Fourier transform infrared (FTIR), electron paramagnetic resonance (EPR), and UV-VIS absorption spectroscopy and by molecular modeling calculations. Distal histidine variants (His64Thr, His64Ile, His64Lys) and charged surface variants (Val67Arg, Lys45Glu, Lys45Glu/Lys63Glu) were included in this study. All variants, with the exception of Val67Arg, have a lower azide affinity than the wild-type protein. Analysis of the temperature dependence of the FTIR spectra (277-313 K) revealed that the wild-type protein and all variants exhibit a high-spin/low-spin equilibrium. Introduction of positively charged amino acid residues shifts nu max for the low-spin form to higher energy while negatively charged residues shifted this maximum to lower energy. The low azide binding affinity exhibited by the His64Thr and His64Ile variants is accompanied by a shift of the nu max for the low-spin infrared band to lower energy and by a significant increase in the corresponding half-bandwidths. This observation indicates greater mobility of the bound azide ligand in these variants. The His64Lys variant exhibits two infrared bands attributable to low-spin forms that are assigned to two different conformations of the lysyl residue. In one conformation, the lysine is proposed to form a hydrogen bond with the bound azide similar to that proposed to occur between the distal histidine and bound azide, and in the other conformation no interaction occurs.

Crystal structures of manganese- and cobalt-substituted myoglobin in complex with NO and nitrite reveal unusual ligand conformations

“…Hoffman and Gibson reported anomalous binding of azide to Mn III Mb, and determined that the reaction of azide with Mn III Mb proceeded in two kinetically separable steps to eventually generate Mn III MbðN À 3 Þ [35]. In contrast, azide binding to iron-containing metMb is consistent with a single binding equilibrium [77]. The 2.8 Å resolution crystal structure of the azide complex of the iron-containing horse metHb is known [78,79].…”

“…The proximal Mn-N(His93) bond length in Mn III ðN À 3 Þ of 2.50 Å is, however, longer than that observed in the iron-containing Mb III ðN À 3 Þ (2.06 Å ), demonstrating the effect that the Mn III d 4 center has on this axial bond length when compared with the ferric d 5 center. Ferric Mb III ðN À 3 Þ has been shown to exhibit a ground-state lowspin electronic configuration and an observable low-spin/ high-spin equilibrium at room temperature [77]. In contrast, the d 4 complex Mn III MbðN À 3 Þ is known to be highspin [35], and this likely accounts for its longer proximal metal-N(His93) bond length.…”

Covalent heme attachment in Synechocystis hemoglobin is required to prevent ferrous heme dissociation

“…Midpoint potentials were extracted from the change in absorbance by fitting to the following equation (Bogumil et al 1994):…”

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]). …”