Henry, Rousseau, Hopfield, Noble and Simon (Biochemistry, 1985) used resonance Raman, optical absorption, and circular dichroism spectroscopy (differential absorption of left- and right-circularly polarized light, which reports on the asymmetric environment of the heme) to show that binding of the allosteric effector inositol hexaphosphate (IHP) — a small molecule that binds in the central cavity of hemoglobin, away from the heme sites, and shifts the protein's quaternary equilibrium — to ferric human and carp hemoglobins shifts the heme-iron spin-state equilibrium toward the high-spin state, with an IHP-induced free-energy change of about 300 cal/mol heme in human derivatives that undergo the quaternary change and about 600 cal/mol in carp derivatives, while the iron position relative to the heme plane is not significantly displaced. The unresolved question they left is the molecular mechanism of this protein work term: whether it is transmitted through protein-induced reorientation of the proximal histidine, through tertiary-structure changes in the heme pocket that electronically perturb the porphyrin macrocycle, or through a specific combination of the two. Follow-up work through 1991 quantified the T-state populations of low-spin derivatives and built a temperature-dependent framework (magnetic-susceptibility measurement with superconducting instrumentation, variable-temperature optical analysis), but no study decomposed the IHP-induced spin free-energy change into enthalpic and entropic parts, established quantitative geometric bounds on the iron and Fe–His geometry, or discriminated the two candidate molecular mechanisms for the ferric spin equilibrium.

Background

Hemoglobin is a four-subunit protein whose cooperative oxygen binding is regulated by an equilibrium between two quaternary structures (the relative arrangement of the four subunits): the T (tense, low-affinity, deoxy-like) and R (relaxed, high-affinity, liganded) states. Methemoglobin (metHb) is the ferric (Fe3+^{3+}) form, unable to bind oxygen; its iron is six-coordinate with a water or exogenous anionic ligand on the distal side and the invariant proximal histidine F8 (position 8 of the F helix, the heme-proximal helix of each globin subunit) on the other side. The ferric iron exists in a thermal spin equilibrium between a high-spin state (S=5/2S=5/2) and a low-spin state (S=1/2S=1/2), whose populations are reported by the intensities of characteristic resonance Raman lines in the 1450–1650 cm−1^{-1} region (a vibrational technique in which laser light tuned to an electronic absorption band selectively enhances vibrational modes of the chromophore) and by the paramagnetic susceptibility (the magnetization of the unpaired iron d electrons, which differs strongly between the two spin states). Inositol hexaphosphate (IHP) is the strongest of the organic-phosphate allosteric effectors — molecules that bind away from the heme sites and shift the R/T equilibrium; it binds in the central cavity of hemoglobin between the two beta subunits and, in ferric derivatives, can drive the T-state quaternary transition. Perutz and coworkers had shown spectroscopically (Perutz, Heidner, Ladner, Beetlestone, Ho and Slade, Biochemistry, 1974) that allosteric transitions shift ferric heme spectra, and Messana, Cerdonio, Shenkin, Noble, Fermi, Perutz and Perutz (Biochemistry, 1978) used superconducting susceptometry (magnetic-susceptibility measurement with a superconducting magnetometer) between 90 and 300 K to show that in carp azide, thiocyanate, and nitrite methemoglobins the thermal spin equilibrium is biased toward high spin by about 1 kcal/mol in the T structure relative to the R structure. Henry, Rousseau, Hopfield, Noble and Simon (Biochemistry, 1985) then made the systematic single-temperature comparison across carp and human metHb derivatives (F−\mathrm{F^-}, H2O\mathrm{H_2O}, SCN−\mathrm{SCN^-}, NO2−\mathrm{NO_2^-}, N3−\mathrm{N_3^-}, CN−\mathrm{CN^-}): Raman spin markers agree with magnetic susceptibility, IHP shifts the spin equilibrium toward high spin (about 300 cal/mol heme in human derivatives that change quaternary structure, about 600 cal/mol in carp), the Fe–CN stretching frequency is unchanged when the quaternary structure changes, and the overall UV and circular dichroism changes track the quaternary transition. They concluded that the IHP-induced protein forces do not significantly move the iron relative to the heme plane, and that the spin shift may instead arise from protein-induced changes in the orientation of the proximal histidine F8 or from tertiary-structure changes in the heme pocket that perturb the porphyrin macrocycle, either of which alters the iron d-orbital energies and hence the spin-state energetics. The related question of how much strain the T state stores in the Fe–imidazole linkage was contested in the ferrous deoxy protein: Kincaid, Stein and Spiro (PNAS, 1979) found heme-imidazole Raman frequencies insensitive (within 2 cm−1^{-1}) to the R-to-T switch of Hb Kempsey, bounding the stored energy below about 0.2 kcal/mol, while Kitagawa and Nagai (in Hemoglobin and Oxygen Binding, 1982) reported resonance Raman evidence for a stretched Fe–Nε\varepsilon(His F8) bond in T-structure deoxyhemoglobin; the 1.74 Å crystal structure of deoxy (T) human hemoglobin by Fermi, Perutz, Shaanan and Fourme (J. Mol. Biol., 1984) provided the geometric reference for the proximal linkage. What remained unresolved in 1985 — and no subsequent study has supplied a definitive published answer — is the molecular mechanism of the IHP-induced spin-state stabilization in the ferric protein: whether it is carried by protein-induced reorientation of the proximal histidine, by tertiary-structure-induced electronic perturbation of the porphyrin, or by a specific combination of the two.

Problem Statement

For ferric human and carp hemoglobins, determine the mechanism by which binding of inositol hexaphosphate (IHP) — and the associated T-state quaternary/tertiary changes — stabilizes the high-spin relative to the low-spin state of the heme iron. Concretely, for the derivatives in which IHP induces the quaternary transition — the F−\mathrm{F^-}, H2O\mathrm{H_2O}, and SCN−\mathrm{SCN^-} derivatives of the human protein (about 300 cal/mol heme of spin free-energy change) and all six carp derivatives studied, F−\mathrm{F^-}, H2O\mathrm{H_2O}, SCN−\mathrm{SCN^-}, NO2−\mathrm{NO_2^-}, N3−\mathrm{N_3^-}, and CN−\mathrm{CN^-} (about 600 cal/mol heme) — identify whether the spin-state shift is transmitted primarily through protein-induced reorientation of the proximal histidine F8, through tertiary-structure changes in the heme pocket that electronically perturb the porphyrin macrocycle, or through a specific combination of the two, and identify which pathway, or combination of pathways, dominates the IHP-induced high-spin stabilization in these derivatives. The mechanism is bounded by the conditions established by the 1985 study at its stated level of precision: the IHP-induced protein forces do not significantly displace the iron atom relative to the heme plane, and the Fe–CN stretching frequency is unchanged upon the quaternary transition.

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