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X-ray analysis of the natural valency hybrid a2+M BostonB32deoxy shows that the ferric iron atoms in the abnormal a subunits are bonded to the phenolate side chains of the tyrosines that have replaced the distal histidines; the iron atoms are displaced to the distal side of the porphyrin ring and are not bonded to the proximal histidines. The resulting changes in tertiary structure of the a subunits stabilize the hemoglobin tetramer in the quaternary deoxy structure, which lowers the oxygen affinity of the normal ,3 subunits and causes cyanosis. The strength of the bond from the ferric iron to the phenolate oxygen appears to be the main factor responsible for the many abnormal properties of hemoglobin M Boston.Hemoglobin (Hb) M Boston is a variant in which the distal histidines in the heme pockets of the a chains are replaced by tyrosines [His E7(58) --Tyr] (1-3). So far it has been found only in heterozygotes, in whom it causes methemoglobinemia and cyanosis. The former is due to the low redox potential of the hemes in the abnormal a chains, which inhibits their reduction by methemoglobin reductase, and the latter to the low oxygen affinity of the hemes in the normal , chains. We have investigated Hb M Boston by x-ray analysis. A difference electron density map of deoxyHbM Boston (a2+B#2) minus A shows that the ferric iron atoms in the a chains are coordinated to four nitrogens of the porphyrin and the phenolate of the tyrosine. The bond to the proximal histidine (F8) is absent and the iron lies on the distal side of the porphyrin ring. The rupture of the iron histidine bond causes the helix F to coil up a little, like a spring released from a tension that had kept it slightly uncoiled. The resulting changes in tertiary structure of the a chain have a stabilizing effect on the quaternary deoxy or T structure of the hemoglobin tetramer, which lowers the oxygen affinity of the normal 3 subunits. EXPERIMENTAL METHODS AND RESULTSThe Hb M Boston used in these studies was identified in a Boston reduces the heme irons in the normal j3, but not in the abnormal a subunits. We were therefore able to crystallize the hybrid a2+B#32 as described for deoxyHb A and found the crystals to be isomorphous with those of A (4). We measured the intensities of about 14,000 reflections within the limiting sphere of 3.5 A-I and calculated a difference Fourier synthesis using (IFBostonI -IFAI) as coefficients, together with the phase angles of deoxyHb A determined by Muirhead and Greer (5) . Fig 1 shows a superposition of several sections through the electron density map of deoxyHb A, comprising the heme iron and the distal histidine of the a subunits. In deoxyHb A the iron atom is displaced by 0.75 A from the plane of the porphyrin ring towards the proximal histidine (6). The difference map shows a large negative peak (b) superimposed on, and slightly to the right of, the iron peak; this is flanked on the left by an even larger positive peak (a), indicating a displacement of the iron atom from right to left. The difference Four...

Multiple internal reflectance infrared (IR) spectra are reported for variably hydrated films (1.2-0.1 g of H2O/g of protein) of the carbon monoxy and oxy forms of human Hb and sperm whale Mb. The spectra show that even the limited removal of liquid and icelike hydration constraints at the globin surface is sufficient to cause a dramatic, but completely reversible, shift toward a normally minute population of sterically unhindered, linear-perpendicular, Fe-CO conformer modes (nu CO = 1968-1967 cm-1), and the destabilization of distally hindered, tilted (or bent), Fe-CO modes (nu CO = 1951, 1944-1933 cm-1). Corroborative evidence from IR band broadening trends [delta delta nu 1/2 (1968, 1967 cm-1) approximately 2-4 cm-1], corresponding changes in the visible, and H-D exchange kinetics confirm that the shift toward 1968-1967 cm-1 results in a more open distal heme pocket configuration and that it is also accompanied by a buildup of deoxy-like steric hindrance proximal to the heme. Denaturation effects are eliminated as a potential cause of the shifts, as are specific protein-protein, ion-protein, intersubunit, and MIR crystal-film surface interactions. The hydration effect exhibits globin-dependent and ligand-dependent differences, which highlight the intrinsic importance of distal steric effects within the heme pocket and their dynamic coupling with exterior solvent constraints. CO-photodissociation and O2-exchange experiments conducted on rapidly interconverting (coupled and fully hydrated) and noninterconverting (uncoupled and partially hydrated) Fe-CO conformers also suggest that the open linear-perpendicular mode corresponds to a more tightly bound form of CO than the axially distorted Fe-CO species; similar differences are not evident in Fe-O2, which already prefers a bent end-on geometry within the heme pocket. Control IR spectra aimed at monitoring the progressive effects of various denaturants on HbCO further indicate that this same open mode serves as a common precursor to any of a number of more highly disordered folding modes. The overall properties of the 1968-1967-cm-1 conformer are discussed in terms of (1) the possibility of its corresponding to an available relaxation mode capable of facilitating the dynamics of ligand entry-release events and (2) its potential additional significance as a native folding mode that exhibits a marked tendency to be destabilized by hydration.

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The 2-(a-hydroxyethyl)-3,4-dimethylthiazolium ion is one of several models for 2-(a-hydroxyethyl)thiamine that were used in elucidating the mechanism of thiamine catalysis. The crystal structures of the title compounds were determined from X-ray diffraction data in order to (1) identify the characteristic steric features of the 2-(a-hydroxyethyl)thiazolium moiety and (2) examine the influence exerted on the thiamine structure when substituents of active intermediates are present at the C(2) position. Both structures were determined from diffractometer data using the 8-28 scan technique with Cu Ka radiation. The structures were refined by the fullmatrix least-squares method. The model compound crystalli5es in the monoclinic space group, P21/c, with unit cell parameters a = 9.771 ( 4 ) , b = 11.656 (3), c = 8.764 (2) A, p = 90.77 (4)O, and 2 = 4. The refinement of this structure converged to a conventional R factor of 0.049 over the 1439 independent observed reflections. The 2-(a-hydroxyethyl)thiamine chloride hydrochloride crystalgzes in the triclinic space group, PT, with unit cell parameters a = 12.811 (3), b = 10.749 (3), c = 7.108 (7) A, a = 108.43 (7), p = 99.05 (7), y = 96.02 (7)O, and 2 = 2. The refinement converged to a conventional R factor of 0.045 based on the 2823 observed reflections. A simple valence bond description of the resonance in the planar thiazolium ring is given which is consistent with the endocyclic bond lengths in both structures. It indicates that there is a partial positiye charge of = on the S atom. The observed intramolecular S . . .O interaction in both compounds, which is 0.4 A less than the normal van der Waals separation, provides evidence that the S bonds electrostatically with the unshared electrons on 0. The conformation of the 2-(a-hydroxyethyl) side chain appears to be strongly infiuenced by the S . e .O interaction. It is remarkably similar in the two compounds. The 0(2a)-C(2a)-C(2)-S(l) torsion angles agree to within 2.2O. The H(2)-0(2a>-C(2a)-C(2) torsion angles both deviate from 90' by less than 12' with the result that they both form a hydrogen bond which is nearly perpendicular to the plane of the thiazolium ring. The analysis of the thiamine derivative shows in addition that its conformation differs considerably from that of thiamine particularly with respect to the relative orientation of the pyrimidine and thiazolium rings. These conformational differences explain the reported shifts in the nmr spectra for these compounds in aqueous solution. On the basis of structural and spectral data, the thiamine and the 2-(a-hydroxyethyl)thiamine molecules may reasonably be expected to assume preferred conformations in aqueous solution closely resembling those in the solid state.uestions concerning details in the three-dimensional Q structure of 2-(a-hydroxyethyl)thiamine (1) have been raised in several papers dealing with the mechanism of thiamine c a t a l y s i~.~-~ In particular, the discussions on possible mechanistic roles for the 4'amino group have lead directly to specu...