Analysis of the contribution of the globin and reductase domains to the ligand-binding properties of bacterial haemoglobins

Abstract

Bacterial Hbs (haemoglobins), like VHb (Vitreoscilla sp. Hb), and flavoHbs (flavohaemoglobins), such as FHP (Ralstonia eutropha flavoHb), have different autoxidation and ligand-binding rates. To determine the influence of each domain of flavoHbs on ligand binding, we have studied the kinetic ligand-binding properties of oxygen, carbon monoxide and nitric oxide to the chimaeric proteins, FHPg (truncated form of FHP comprising the globin domain alone) and VHb-Red (fusion protein between VHb and the C-terminal reductase domain of FHP) and compared them with those of their natural counterparts, FHP and VHb. Moreover, we also analysed polarity and solvent accessibility to the haem pocket of these proteins. The rate constants for the engineered proteins, VHb-Red and FHPg, do not differ significantly from those of their natural counterparts, VHb and FHP respectively. Our results suggest that the globin domain structure controls the reactivity towards oxygen, carbon monoxide and nitric oxide. The presence or absence of a reductase domain does not affect the affinity to these ligands.

Figure 1. Haem staining

Purified Hb proteins were separated by SDS/PAGE and stained with TMB for haem detection. Lane 1, molecular-mass markers; lane 2, VHb-Red; lane 3, VHb-Red; lane 4, VHb; lane 5, VHb-Red; lane 6, FHP; and lane 7, FHPg.

Figure 2. FHPg spectra

Black line, purified protein, oxidized form; grey line, purified protein in the presence of sodium dithionite, partially reduced form.

Figure 3. O₂, CO and NO recombination kinetics after flash photolysis

(A) Time courses for O₂ rebinding to bacterial Hbs and flavoHbs after flash photolysis in the presence of 650 μM O₂. (B) Time courses for CO rebinding to bacterial Hbs and flavoHbs after flash photolysis in the presence of 500 μM CO. (C) Time courses for NO rebinding to Fe(III) form of bacterial Hbs and flavoHbs after flash photolysis in the presence of 400 μM NO. To facilitate comparison of the data for the various proteins, the absorbance changes were normalized to 1 at the maximum absorbance. Line 1, VHb; line 2, VHb-Red; line 3, FHP; line 4, FHPg. Protein concentrations were 2–10 μM in 0.1 M sodium phosphate buffer (pH 7.0), The temperature was 20 °C.

Figure 4. Haem pocket opening of FHP and FHPg

The structure on the left is FHP (1cqx); that on the right is FHPg (1cqx, residues 1-147). The Figure shows the globin domain (magenta), the reductase domain (ice blue), the haem molecule (red), FAD (yellow) and lipid (green).