Quaternary structures of intermediately ligated human hemoglobin a and influences from strong allosteric effectors: resonance Raman investigation

Abstract

The Fe-histidine stretching (nu(Fe-His)) frequency was determined for deoxy subunits of intermediately ligated human hemoglobin A in equilibrium and CO-photodissociated picosecond transient species in the presence and absence of strong allosteric effectors like inositol(hexakis)phosphate, bezafibrate, and 2,3-bisphosphoglycerate. The nu(Fe-His) frequency of deoxyHb A was unaltered by the effectors. The T-to-R transition occurred around m = 2-3 in the absence of effectors but m > 3.5 in their presence, where m is the average number of ligands bound to Hb and was determined from the intensity of the nu(4) band measured in the same experiment. The alpha1-beta2 subunit contacts revealed by ultraviolet resonance Raman spectra, which were distinctly different between the T and R states, remained unchanged by the effectors. This observation would solve the recent discrepancy that the strong effectors remove the cooperativity of oxygen binding in the low-affinity limit, whereas the (1)H NMR spectrum of fully ligated form exhibits the pattern of the R state.

FIGURE 1

The 441.6-nm excited RR spectra of deoxyHb at pH 6.3 (BZF 0.5 mM and IHP 2 mM, A), at pH 6.3 (BZF 5mM, B), at pH 7.5 (BPG 2 mM, C), and at pH 8.9 (no effector, D). All samples are equilibrated with 0.05 M HEPES buffer, containing 0.1 M Na₂SO₄, and protein concentrations were 200 μM in heme. Laser power at the sample point was 2.1 mW and the spectra are the sum of 20 exposures of 60 s each.

FIGURE 2

The 442-nm excited RR spectra at 10 ps after photodissociation of COHb upon pumping at 540 nm for the solution conditions of pH 6.5, with 0.75 mM BZF and 3 mM IHP (A), pH 6.5 with 7.5 mM BZF (B), pH 7.4 with 3 mM BPG (C), and pH 8.8 with no effectors (D). All samples are equilibrated with 0.05 M HEPES buffer, containing 0.1 M Na₂SO₄, and protein concentrations were 300 μM in heme. Panel a shows the spectral region between 150 and 1050 cm⁻¹,, whereas panel b shows their expansion for the frequency region between 190 and 330 cm⁻¹. The spectral contributions from nonphotodissociated species have been subtracted.

FIGURE 3

The 235-nm excited UVRR spectra of deoxyHbA at pH 6.6 with 1 mM BZF and 5 mM IHP (A), and at pH 7.5 with 5 mM BPG (B), and their respective deoxyHb-minus-COHb difference spectra. D and F are difference spectra for A and B, respectively, whereas E is the corresponding difference for the solution at pH 6.4 with 5 mM IHP and G denotes the corresponding difference of the isolated α chain in the presence of 5 mM IHP and 1 mM BZF. The 235-nm excited UVRR spectrum of BZF (1 mM) in the same buffer is also included as spectrum C. The assignments of bands are marked beside the wavenumber, in which Y and W denote the modes of Tyr and Trp residues, respectively. The band at 980 cm⁻¹ arises from ions contained as an internal intensity standard. All samples of Hb are equilibrated with 0.05 M HEPES buffer, containing 0.2 M Na₂SO₄, and the protein concentrations were 400 μM in heme.

FIGURE 4

(a) The 441.6-nm excited RR spectra of COHb in a steady state at pH 6.6 with 1 mM BZF and 5 mM IHP, measured with a spinning cell of varied rates: (A) 1800 rpm; (B) 600 rpm; and (C) 180 rpm. Laser power used was 3.0, 0.37, and 0.027 mW for A, B, and C, respectively. (b) The 441.6-nm excited RR spectra of fully deoxyHbA and fully CO-bound HbA at pH 8.8 with no effector, measured with a spinning cell of 1800 rpm. The two spectra were observed under the same experimental conditions with a laser power of 110 μW at the sample point. All samples are equilibrated with 0.05 M HEPES buffer, containing 0.1 M Na₂SO₄, and protein concentrations were 200 μM in heme. (c) Simulated ν₄ bands for different numbers (m = 0–4) of CO molecules bound to Hb, which were calculated from the spectra shown in the middle panel.

FIGURE 5

The 441.6-nm excited RR spectra of COHb for a certain fixed number of CO molecules bound to Hb under solution conditions 5 and 1. The spinning rate of the cell is 1800 rpm (A), 600 rpm (B), and 180 rpm (C) for solution 5 and 180 rpm for solution 1 (D). All samples are equilibrated with 0.05 M HEPES buffer, containing 0.1 M Na₂SO₄. Protein concentrations were 200 μM in heme.

FIGURE 6

The 441.6-nm excited RR spectra of COHb in solution conditions 1 (a) and 5 (b) observed with different laser powers under constant spinning rate (180 rpm), except for A and A′, which represent the spectra of deoxyHb. The m value for each spectrum of solution 1 in panel a is as follows: 1.3 (B), 1.7 (C), 2.1 (D), 2.8 (E), 3.3 (F), 3.6 (G), and 3.9 (H). The m value of COHb for each spectrum in solution 5 in panel b in the presence of IHP and BZF at pH 6.6 is as follows: 2.9 (B′), 3.5 (C′), and 3.8 (D′). The m values were determined with the corresponding ν₄ spectra in the inset figures and the calibration curves shown in Fig. 4 c. All samples are equilibrated with 0.05 M HEPES buffer, containing 0.1 M Na₂SO₄. Protein concentrations were 200 μM in heme.

FIGURE 7

Schematic free energy diagram of Hb for oxygen binding. Gibbs free energy level of T₀–T₄ and R₀–R₄ are represented by horizontal bars, in which subscripts denote the m value. It is assumed that the T₀–T₄ system is changed relative to the R₀–R₄ system from black to gray by the change of solvent conditions from 1 to 5. When T₃ is higher than R₃ (black lines), the intersystem crossing occurs in the T₂-to-R₃ transition. When T₃ is lower than R₃ (shaded lines), the intersystem switching occurs between T₃ and R₄, and as a result, cooperativity is apparently lost.