Quaternary Structure and Spin Equilibria in Ferric Hemoglobins

Abstract

The effective magnetic moments for a number of human and carp methemoglobin derivatives were determined in solution at room temperature. The data permit us to confirm the dependence of the spin-state equilibrium of azide methemoglobin on the quaternary state of the hemoglobin and to demonstrate a similar dependence for both human and carp aquomethemoglobin.In addition, the pH dependence of the effective magnetic moment and the Soret spectrum of carp azidemethemoglobin are compared.In 1978 Perutz et al. [I] and Messana et al. [2] reported that the spin-state equilibria of derivatives of ferric hemoglobin were thermodynamically linked to the conformational equilibrium of the quaternary structure of the protein. This was most clearly demonstrated by studying the effect of the conversion of carp azidemethemoglobin from the R to the T quaternary state on the equilibrium between low-spin and high-spin forms of the derivative. Indications of the shift in spin-state equilibrium were found by a variety of spectroscopic techniques, but the most direct demonstration was obtained by measurements of magnetic susceptibility. These authors measured the molar magnetic susceptibilities of a number of ferric derivatives of carp and human hemoglobin in the R and T state. At that time the method for determination of the molar paramagnetic contribution to the susceptibility relied in part on measurements over an extended temperature interval in the frozen state. It is now possible to estimate the paramagnetic contribution to susceptibility in the liquid state without examining the temperature dependence. This allows us to define the properties of the system at physiological temperature with much greater precision than was previously possible. In addition, this method permits the examination of the effects of solution variables, such as pH, which are difficult to define in frozen systems.We furthermore wanted to reexamine the effect of quaternary structure on the magnetic properties of aquomethemoglobin. Perutz et al. reported [l] that the magnetic susceptibility of this hemoglobin derivative was unaffected by changes in the quaternary structure from R to T state. At the time, this finding was unexpected in view of the very significant change in the visible spectrum of this derivative, as a consequence of the conformational transition. This spectral change strongly suggested a change in spin-state equilibrium. In addition, recent reasonance Raman spectroscopic studiesAbbreviations. R, relaxed (high-affinity) state of hemoglobin; T, tense (low-affinity) state of hemoglobin; NMR, nuclear magnetic resonance; P6-inosito1, inositol hexakisphosphate; pB, Bohr magneton.of the ferric derivatives of carp and human hemoglobins carried out in the laboratory of Dr Dennis Rousseau suggest that in fluoromethemoglobin the R and T transition has no effect on spin-state equilibrium, but that for aquomethemoglobin a small but significant change is observed (E. R. Henry, D. Rousseau, S. Simon, J. J. Hopfield, and R. W. Noble, unpublished results). Si...

Quaternary structure has little influence on spin states in mixed-spin human methemoglobins

“…More attention has been focused on carp metHbs, which show larger effects of IHP. For carp azide metHb, the data are consistent with a value of ~1 kcal/mol for AAG(spin) (Perutz et al, 1978;Messana et al, 1978;Noble et al, 1983).…”

“…There have been several studies of the influence of changes in quaternary structure on the spin equilibria of metHbs, both by direct magnetic measurements and by indirect spectroscopic evidence. In nearly all of these studies, inositol hexaphosphate (IHP), a powerful allosteric effector that binds preferentially to the T structure, has been used to switch the quaternary structure from R to T. The results for human metHbs are conflicting and have not been calibrated in terms of energy but generally indicate that addition of IHP produces only small shifts of the spin equilibria toward the high-spin state (Perutz et al, 1974(Perutz et al, , 1978; Gupta & Mildvan, 1975;Messana et al, 1978;Noble et al, 1983). More attention has been focused on carp metHbs, which show larger effects of IHP.…”

Spin equilibrium in human methemoglobin: effects of inositol hexaphosphate and bezafibrate as measured by resonance Raman spectroscopy

“…When both IHP and BZF are added to the mixed-spin derivatives (H20, SCN", OCN", and N02") of human methemoglobin, the spin equilibrium is shifted toward higher spin by about 700 cal/mol, similar to the spin change detected in derivatives of carp methemoglobin upon addition of IHP alone. These data support a general mechanism for the allosteric transition in which a constant fraction of the cooperative energy («20%) is detected at the heme of the ferric ligand-bound forms.e spin equilibrium in methemoglobins has been studied extensively to monitor the influence of protein structural changes on the heme group (Perutz et al, 1978;Noble et al, 1983;Henry et al, 1985). The addition of organic phosphates to the protein has been shown to alter this equilibrium and in some cases to bring about a change in the quaternary…”

“…e spin equilibrium in methemoglobins has been studied extensively to monitor the influence of protein structural changes on the heme group (Perutz et al, 1978;Noble et al, 1983;Henry et al, 1985). The addition of organic phosphates to the protein has been shown to alter this equilibrium and in some cases to bring about a change in the quaternary…”

Bonding of heme FeIII with dioxygen: observation and characterization of an incipient bond

“…Note that here the spin equilibrium is unaffected by the presence of a surrounding protein. 13 Bunches of ions are extracted from a first octopole trap and mass-selected by a pulsed mass-gate during their flight into a quadrupole ion trap maintained at the desired temperature (10-100 K) using a cryostat. These experiments have been performed on a new experimental setup of the CLUPS facility (Orsay, France).…”