MODELING THE HEMOGLOBIN SWITCHPOINT WITH CYANOMET VALENCY HYBRIDS - RAMAN-SPECTROSCOPIC PROBES OF TERTIARY AND QUATERNARY STRUCTURE

Hybrid hemoglobins with cyanomet hemes in the alpha or in the beta cha ins have been investigated by resonance Raman (RR) spectroscopy, using ultraviolet (230 nm) and visible (441.6 nm) excitation. For the CO ad ducts, the UVRR spectra are identical with that of native HbCO, showin g the tyrosine and tryptophan signals to be insensitive to ligand subs titution within the R state. In the absence of CO, the doubly ligated hybrids show differences in the UVRR spectra, relative to the CO adduc ts, which are the superposition of two difference spectra: (1) the T-R difference spectrum obtained by subtracting the spectrum of HbCO from that of deoxyHb and (2) a perturbed R state spectrum, characteristic of deligated chains within the R state. These spectra arise from alter ations, respectively, in the quaternary contacts of interface aromatic residues and in the tertiary contacts of interior aromatic residues. From the amplitudes of the difference spectra, the T state population was determined to be 30% for (alpha(FeII)beta(FeIII)CN)(2) and 43% for (alpha(FeIII)CN beta(FeII))(2), in good agreement with the kinetic an alysis of Cassoly and Gibsoin [Cassoly, R., & Gibson, Q. H. (1972) J. Biol. Chem. 247, 7332]. Addition of inositol hexaphosphate (IHP) incre ased the T state population, but only by a modest amount, to 40 and 53 %, respectively, in contrast to the frequent assumption that the T sta te conversion is quantitative in the presence of IHP. Since current un derstanding of the quaternary state dependence of the Fe-histidine str etching frequency is based on that assumption, the RR band envelope fo r this vibration was reexamined. For (alpha(FeII)beta(FeIII)CN)(2) th, band is readily resolvable into two components, whose areas yield the same T/R population ratios as do the UVRR difference spectra, in the presence and in the absence of IHP. The resolved components yield 223 and 210 cm(-1) as the R and T state frequencies; they both shift signi ficantly upon IHP binding, to 219 and 203 cm(-1), revealing an IHP eff ect on tertiary as well as quaternary structure. For (alpha(FeIII)CN b eta(FeII))(2), the components are not independently resolvable, but fi xing their areas to correspond to the UVRR-derived T/R ratios yielded R and T state frequencies of 222 and 213 cm(-1), with no perceptible s hift upon IHP binding. The T/R ratios are much lower than those derive d from oxygenation curves for CN- adducts of partially oxidized Hb [Ma rden, M. C., Kiger, L., Kister, J., Bohn, B., & Poyart, C. (1991) Biop hys. J. 60, 770]. This apparent discrepancy is ascribed to a preponder ance of asymmetric hybrids (alpha(1)(FeII)beta(1)(FeIII)alpha(2)(FeIII )CN beta(2)(FeIII)CN), which Ackers et al. [Ackers, G. K., Doyle, M. L ., Myers, D., & Daugherty, M. A. (1992) Science 255, 54] have shown to be thermodynamically more stable than the symmetric hybrids. The R st ate perturbation for unligated chains which is revealed in the UVRR sp ectra is believed to involve collapse of the E helix toward the heme a nd weakening of a tryptophan hydrogen bond between the E and A helices . This perturbation is suggested to account for the phenomenon of quat ernary enhancement, an R state augmentation of ligand affinity for unl igated chains.