Near-UV Circular Dichroism and UV Resonance Raman Spectra of Individual Tryptophan Residues in Human Hemoglobin and Their Changes upon the Quaternary Structure Transition

The aromatic residues such as tryptophan (Trp) and tyrosine (Tyr) in human adult hemoglobin (Hb A) are known to contribute to near-UV circular dichroism (CD) and UV resonance Raman (RR) spectral changes upon the R → T quaternary structure transition. In Hb A, there are three Trp residues per αβ dimer: at α14, β15, and β37. To evaluate their individual contributions to the R → T spectral changes, we produced three mutant hemoglobins in <i>E. coli</i>; rHb (α14Trp→Leu), rHb (β15Trp→Leu), and rHb (β37Trp→His). Near-UV CD and UVRR spectra of these mutant Hbs were compared with those of Hb A under solvent conditions where mutant rHbs exhibited significant cooperativity in oxygen binding. Near-UV CD and UVRR spectra for individual Trp residues were extracted by the difference calculations between Hb A and the mutants. α14 and β15Trp exhibited negative CD bands in both oxy- and deoxy-Hb A, whereas β37Trp showed positive CD bands in oxy-Hb A but decreased intensity in deoxy-form. These differences in CD spectra among the three Trp residues in Hb A were ascribed to surrounding hydrophobicity by examining the spectral changes of a model compound of Trp, <i>N</i>-acetyl-l-Trp ethyl ester, in various solvents. Intensity enhancement of Trp UVRR bands upon the R → T transition was ascribed mostly to the hydrogen-bond formation of β37Trp in deoxy-Hb A because similar UVRR spectral changes were detected with <i>N</i>-acetyl-l-Trp ethyl ester upon addition of a hydrogen-bond acceptor

Left: The CD spectral changes due to the quaternary and tertiary structure transition for Hb A.

<p>The spectra are the quaternary structure transition (A: pink spectrum) and tertiary structure transition (B: blue spectrum) expected for Hb A and the observed deoxy-minus-oxy difference spectra of Hb A (C: black spectrum). <b>Right: Comparison of the deoxy-minus-oxy difference spectra of rHb(αH87G) and rHb(βH92G) with that of Hb A.</b> The difference spectra are Hb A (black spectrum), rHb(αH87G) (orange spectrum) and rHb(βH92G) (green spectrum).</p

Hill’s plots of oxygen binding by Hb A, rHb(αH87G), rHb(βH92G) and sperm whale Mb.

<p>Hb A (-IHP) (blue closed triangle: ▲), Hb A (+IHP) (pink closed square: ■), rHb(αH87G) (black closed circle: ●), rHb(βH92G) (orange closed circle: ●) and sperm whale Mb (light green closed diamond: ◆). <i>Y</i> and <i>p</i>O₂ are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135080#pone.0135080.g002" target="_blank">Fig 2</a>. The symbols are the observed points. The hemoglobin concentration was 60 μM on a heme basis in 0.05 M bis-Tris buffer (pH 7.4) containing 0.1 M Cl^-. In addition, rHb(αH87G) and rHb(βH92G) contained 5 mM imidazole and a metHb reducing system. The temperature was set at 25°C. IHP was added to a final concentration of 2 mM.</p

The 229-nm excited UVRR spectra of Hb A, rHb(αH87G) and rHb(βH92G).

<p>Spectra are deoxyHb A (A), deoxy rHb(αH87G) (B) and deoxy rHb(βH92G) (C), and the difference between Hb A (deoxy–CO) (D), rHb(αH87G) (deoxy–CO) (E) and rHb(βH92G) (deoxy–CO) (F). The hemoglobin concentration was 200 μM (in heme) in a 0.05 M phosphate buffer (pH 7.0) containing 0.2 M SO₄^2- as the internal intensity standard. In addition, rHb(αH87G) and rHb(βH92G) contained 10 mM imidazole. The difference spectra were obtained so that the Raman band of SO₄^2- (980 cm^-1) could be abolished. The spectra shown are an average of 13 scans.</p

An Origin of Cooperative Oxygen Binding of Human Adult Hemoglobin: Different Roles of the α and β Subunits in the α₂β₂ Tetramer

<div><p>Human hemoglobin (Hb), which is an α₂β₂ tetramer and binds four O₂ molecules, changes its O₂-affinity from low to high as an increase of bound O₂, that is characterized by ‘cooperativity’. This property is indispensable for its function of O₂ transfer from a lung to tissues and is accounted for in terms of T/R quaternary structure change, assuming the presence of a strain on the Fe-histidine (His) bond in the T state caused by the formation of hydrogen bonds at the subunit interfaces. However, the difference between the α and β subunits has been neglected. To investigate the different roles of the Fe-His(F8) bonds in the α and β subunits, we investigated cavity mutant Hbs in which the Fe-His(F8) in either α or β subunits was replaced by Fe-imidazole and F8-glycine. Thus, in cavity mutant Hbs, the movement of Fe upon O₂-binding is detached from the movement of the F-helix, which is supposed to play a role of communication. Recombinant Hb (rHb)(αH87G), in which only the Fe-His in the α subunits is replaced by Fe-imidazole, showed a biphasic O₂-binding with no cooperativity, indicating the coexistence of two independent hemes with different O₂-affinities. In contrast, rHb(βH92G), in which only the Fe-His in the β subunits is replaced by Fe-imidazole, gave a simple high-affinity O₂-binding curve with no cooperativity. Resonance Raman, ¹H NMR, and near-UV circular dichroism measurements revealed that the quaternary structure change did not occur upon O₂-binding to rHb(αH87G), but it did partially occur with O₂-binding to rHb(βH92G). The quaternary structure of rHb(αH87G) appears to be frozen in T while its tertiary structure is changeable. Thus, the absence of the Fe-His bond in the α subunit inhibits the T to R quaternary structure change upon O₂-binding, but its absence in the β subunit simply enhances the O₂-affinity of α subunit.</p></div

The Hill plot of oxygen binding by rHb(αH87G).

<p>The symbols are the observed points. <i>Y</i> is the fractional oxygen saturation and <i>p</i>O₂ is the partial pressure of oxygen in millimeters of Hg. Asymptotic lines in the high and low affinity subunits are drawn by eye, and correspond to the <i>K</i>₁ and <i>K</i>₄ values of the four stepwise Adair constants, respectively. The hemoglobin concentration was 60 μM on a heme basis in 0.05 M bis-Tris buffer (pH 7.4) containing 0.1 M Cl^-, 5 mM imidazole and a metHb reducing system. The temperature was set at 25°C.</p

600 MHz ¹H NMR spectra of Hb A, rHb(αH87G) and rHb(βH92G).

<p>Spectra are CO- and deoxyHb A (A, B), CO- and deoxy-rHb(αH87G) (C, D), and CO- and deoxy-rHb(βH92G) (E, F) between 10 and 16 ppm at pH 7.0 and 25°C. The hemoglobin concentrations of Hb A, rHb(αH87G) and rHb(βH92G) were 1 mM, 800 and 500 μM, respectively, on a heme basis in 0.05 M phosphate buffer (pH 7.0). In addition, rHb(αH87G) and rHb(βH92G) contained 10 mM imidazole.</p

Possible roles of the Fe-His bonds of α and β subunits on cooperative O₂ binding.

<p>Possible roles of the Fe-His bonds of α and β subunits on cooperative O₂ binding.</p

Schematic presentation of the normal heme (left) and the cavity mutant heme (F8His→Gly) (right).

<p>The crystal structure of the cavity mutant Mb, rMb(H93G), determined in the presence of imidazole, revealed that an imidazole molecule is bonded to the heme iron on the proximal side, as shown here (Barrick, D. <i>et al</i>., <i>Biochemistry</i><b>1994</b>, <i>33</i>, 6546−6554). Atomic coordinates of 2DN2 (ref. 46) were used about description of ribbon model of F-helix.</p

The 441.6-nm excited visible RR spectra of deoxyHb A (A), deoxy-rHb(αH87G) (B) and deoxy-rHb(βH92G) (C).

<p>The hemoglobin concentration was 200 μM (in heme) in a 0.05 M phosphate buffer, pH 7.0. In addition, rHb(αH87G) and rHb(βH92G) contained 10 mM imidazole. For rHb(αH87G), deconvoluted components, a ν_Fe-His and a ν_Fe-Im are indicated by a thin dotted red line and a brown solid line, respectively. For rHb(βH92G), deconvoluted components, a ν_Fe-His (high), a ν_Fe-His (low) and a ν_Fe-Im are indicated by a thin dotted green, a solid blue line and a solid brown line, respectively.</p