Redox Control in Heme Proteins: Electrostatic Substitution in the Active Site of Leghemoglobin

• Published in: Archives of biochemistry and biophysics Vol. 400; no. 1; pp. 111 - 117
• Main Authors: Jones, Deborah K., Patel, Neesha, Lloyd Raven, Emma
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.04.2002
• Subjects: Aspartic Acid - chemistry; Binding Sites; charge substitution; Circular Dichroism; Electrochemistry; Glycine max - metabolism; heme; Heme - chemistry; Hydrogen Bonding; Hydrogen-Ion Concentration; Kinetics; leghemoglobin; Leghemoglobin - chemistry; Leghemoglobin - metabolism; Leucine - chemistry; Models, Chemical; Mutagenesis, Site-Directed; Oxidation-Reduction; Protein Binding; Recombinant Proteins - metabolism; redox; reduction potential; Spectrophotometry; Spectrum Analysis, Raman; Time Factors; leghemoglobin; charge substitution; heme; reduction potential; redox
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.2002.2771

The effects of electrostatic substitutions on the spectroscopic, ligand binding, and redox properties of the heme in leghemoglobin have been examined by replacement of the proximal leucine 88 residue with an aspartic acid residue (Leu88Asp). Electronic and resonance Raman spectra of the ferric derivative of Leu88Asp indicate a mixture of 6-coordinate, high-spin and 6-coordinate, low-spin hemes, analogous to that observed in the recombinant wild-type protein (rLb). At alkaline pH, formation of hydroxide-bound heme is indicated for Leu88Asp; the pKa for this transition (8.7 ± 0.2, μ = 0.10 M, 25.0°C) is 0.4 pH units higher than for rLb. Equilibrium dissociation constants (sodium phosphate, pH 7.0, μ = 0.10 M, 25.0 ± 0.1°C) for binding of anionic ligands (N−3, nicotinate) to Leu88Asp are higher (Kd,nicotinate = 6.8 ± 0.2 μM; Kd,azide = 33 ± 0.6 μM) than the corresponding values for rLb (Kd,nicotinate = 1.4 ± 0.3 μM (pH 5.5, μ = 0.10 M, 25.0 ± 0.1°C); Kd,azide = 4.8 ± 0.2 μM). Resonance Raman spectra (sodium phosphate, pH 7.0, μ = 0.10 M) for the ferrous derivatives of Leu88Asp and rLb exhibit a strong νFe-His stretching frequency at 223 cm−1 in both cases, indicating that the hydrogen bonding structure on the proximal side is not substantially altered in the variant. The reduction potential of Leu88Asp is −14 ± 2 mV vs standard hydrogen electrode (SHE) (25.0°C, μ = 0.10 M, pH 7.0), a decrease of 35 mV over the corresponding value for the wild-type protein under the same conditions (21 ± 3 mV vs SHE). An assessment of these data in terms of electrostatic and hydrogen bonding considerations is presented.