Alteration of sperm whale myoglobin heme axial ligation by site-directed mutagenesis

Abstract

Three mutant proteins of sperm whale myoglobin (Mb) that exhibit altered axial ligations were constructed by site-directed mutagenesis of a synthetic gene for sperm whale myoglobin. Substitution of distal pocket residues, histidine E7 and valine E11, with tyrosine and glutamic acid generated His(E7)Tyr Mb and Val(E11)Glu Mb. The normal axial ligand residue, histidine F8, was also replaced with tyrosine, resulting in His(F8)Tyr Mb. These proteins are analogous in their substitutions to the naturally occurring hemoglobin M mutants (HbM). Tyrosine coordination to the ferric heme iron of His(E7)Tyr Mb and His(F8)Tyr Mb is suggested by optical absorption and EPR spectra and is verified by similarities to resonance Raman spectral bands assigned for iron-tyrosine proteins. His(E7)Tyr Mb is high-spin, six-coordinate with the ferric heme iron coordinated to the distal tyrosine and the proximal histidine, resembling Hb M Saskatoon [His(beta E7)Tyr], while the ferrous iron of this Mb mutant is high-spin, five-coordinate with ligation provided by the proximal histidine. His(F8)Tyr Mb is high-spin, five-coordinate in both the oxidized and reduced states, with the ferric heme iron liganded to the proximal tyrosine, resembling Hb M Iwate [His(alpha F8)Tyr] and Hb M Hyde Park [His(beta F8)Tyr]. Val(E11)Glu Mb is high-spin, six-coordinate with the ferric heme iron liganded to the F8 histidine. Glutamate coordination to the ferric iron of this mutant is strongly suggested by the optical and EPR spectral features, which are consistent with those observed for Hb M Milwaukee [Val(beta E11)Glu]. The ferrous iron of Val(E11)Glu Mb exhibits a five-coordinate structure with the F8 histidine-iron bond intact.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential heme release from various hemoglobin redox states and the upregulation of cellular heme oxygenase‐1

“…These changes include a decrease in intensity, slight red‐shift of the Soret maximum and the appearance of an intense band at 600 nm which gives a green color. The intense absorption at 600 nm is attributed to the direct coordination of the tyrosine phenol side chain to the iron atom 22.…”

Properties of Human Hemoglobins with Increased Polarity in the α- or β-Heme Pocket

“…Contrary to the deoxy derivatives, in which the equilibrium position of the iron atom out of the mean heme plane gives rise to a nonsymmetric inhomogeneous broadening of the Soret band (see Cupane et al (10) for an in-depth discussion), in the CO derivatives, the effect of conformational heterogeneity can be modeled as a gaussian distribution of 0 -0 transition frequencies and gives rise to the additional term in 2 in Equation 4. IR Spectra-Infrared spectra were recorded at room temperature using a Perkin-Elmer 1600 series FT-IR spectrometer.…”

“…The above issues have been extensively investigated using site-directed mutagenesis, especially in myoglobin, which can be considered a prototype of oxygen transport proteins (2). On the distal side, the role of Val(E11), His(E7), Leu(B10), Phe(CD1), and Phe(CD4) has been investigated by replacing these residues primarily with amino acids of different size to investigate the role of steric hindrance in ligand binding (3)(4)(5)(6). These studies indicate that, although steric hindrance is, in certain cases, an effective means of regulating ligand affinity, it is not the major determinant.…”

Catalase Reaction by Myoglobin Mutants and Native Catalase

“…Myoglobin (Mb), 1 a carrier of molecular oxygen, has been studied as a structural and/or functional model for elucidating the role of active site residues in heme enzymes (7)(8)(9)(10)(11)(12)(13)(14). Recently, we have proven that the distal histidine (His-64) in sperm whale Mb is a critical residue in destabilizing Mb compound I (Mb-I).…”