Alteration of sperm whale myoglobin heme axial ligation by site-directed mutagenesis

Three mutant proteins of sperm whale myoglobin (Mb) that exhibit altered axial ligations were constructed by site-directed mutagenesis of a synthetic gene for sperm whale myoglobin. Substitution of distal pocket residues, histidine E7 and valine E11, with tyrosine and glutamic acid generated His(E7)Tyr Mb and Val(E11)Glu Mb. The normal axial ligand residue, histidine F8, was also replaced with tyrosine, resulting in His(F8)Tyr Mb. These proteins are analogous in their substitutions to the naturally occurring hemoglobin M mutants (HbM). Tyrosine coordination to the ferric heme iron of His(E7)Tyr Mb and His(F8)Tyr Mb is suggested by optical absorption and EPR spectra and is verified by similarities to resonance Raman spectral bands assigned for iron-tyrosine proteins. His(E7)Tyr Mb is high-spin, six-coordinate with the ferric heme iron coordinated to the distal tyrosine and the proximal histidine, resembling Hb M Saskatoon [His(beta E7)Tyr], while the ferrous iron of this Mb mutant is high-spin, five-coordinate with ligation provided by the proximal histidine. His(F8)Tyr Mb is high-spin, five-coordinate in both the oxidized and reduced states, with the ferric heme iron liganded to the proximal tyrosine, resembling Hb M Iwate [His(alpha F8)Tyr] and Hb M Hyde Park [His(beta F8)Tyr]. Val(E11)Glu Mb is high-spin, six-coordinate with the ferric heme iron liganded to the F8 histidine. Glutamate coordination to the ferric iron of this mutant is strongly suggested by the optical and EPR spectral features, which are consistent with those observed for Hb M Milwaukee [Val(beta E11)Glu]. The ferrous iron of Val(E11)Glu Mb exhibits a five-coordinate structure with the F8 histidine-iron bond intact.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR characterization of a diamagnetic model of unliganded alpha chains from human hemoglobin.

This paper reports the reconstitution and spectroscopic characterization of a complex between alpha globin from human adult hemoglobin and protoporphyrin IX-Zn(II). Optical and proton one-dimensional (1-D) NMR spectra indicate that the prosthetic group binds in a 1:1 stoichiometry to the apoglobin in a single conformation. Using 2-D proton NMR techniques we assigned resonances corresponding to the majority of porphyrin substituents and to several side chains of amino acids in contact with the porphyrin. Analysis of nuclear Overhauser enhancement interactions between identified protons indicated that the complex contains only one rotation isomer of the prosthetic group. The diamagnetic Zn(II) ion is coordinated to the proximal histidine (His87) and does not bind O2 or CO as a sixth ligand. The ring current effects on protons from the distal valine (Val62) are considerably higher than in the liganded form providing strong evidence for a more compact ligand binding pocket relative to the carbon monoxy state. Therefore, protoporphyrin-Zn(II)/alpha globin complex is a suitable diamagnetic model for unliganded alpha chains and will be used for structure determination by NMR and modeling methods.     

Studies on cobalt myoglobins and hemoglobins, XIII. A consequence of the occurrence of glutamine at the E7 (58) site of alpha subunits in opossum hemoglobin

To investigate the mode of interactions between heme metal, bound oxygen and the distal residue at the E7 site, we have measured accurate oxygen equilibrium curves, oxygen binding relaxations following temperature-jump, and electron paramagnetic resonance spectra of natural and cobalt-substituted opossum hemoglobin, which has glutamine and histidine at the E7 site of the α chain and the β chain, respectively, and compared them with those of natural and cobalt-substituted human hemoglobin, which has histidine at the E7 site of both the α and β chains.Natural opossum hemoglobin has a lower oxygen affinity, slightly smaller and pH-dependent co-operativity, a somewhat greater Bohr effect, and a smaller effect of organic phosphates such as 2,3-diphosphoglycerate and inositol hexaphosphate on oxygen affinity as compared to natural human hemoglobin. Upon substitution of cobalt for iron, these oxygenation characteristics of opossum hemoglobin relative to those of human hemoglobin were preserved well. The behavior of the intrinsic oxygen association constants pertaining to the four oxygenation steps (i.e. the Adair constants) upon addition of the organic phosphates or pH changes indicates that the allosteric equilibrium in opossum hemoglobin is biased towards the T state as compared with that in human hemoglobin, and that the oxygen affinity of the R structure is lower for opossum hemoglobin than for human hemoglobin. The temperature-jump kinetic data indicate that the lower oxygen affinity of opossum cobalt-hemoglobin in comparison with that of human cobalt-hemoglobin can be ascribed to a decreased oxygen association rate constant. The electron paramagnetic resonance experiments on oxy and deoxy opossum and human cobalt-hemoglobins in buffered H₂O and ²H₂O, including their photolysed products at a low temperature, provided the following information. The cobaltous ion of the α subunits of deoxy opossum cobalt-hemoglobin is in an environment that is similar to that for cobaltous ions of deoxy human cobalt-hemoglobin in the T state. The hydrogen bond between the bound oxygen and the residue at E7, which has been shown to exist in oxy human cobalt-hemoglobin and oxy sperm whale cobalt-myoglobin, is absent or, at least, significantly altered in the α subunits of oxy opossum cobalt-hemoglobin, probably resulting in a lower oxygen affinity. Interference by isoleucine at E11α with an oxygen molecule is suggested as an explanation for the lowered affinity of opossum iron-hemoglobin. However, no straightforward structural explanation is available for the lower oxygen affinity of the R structure and the allosteric equilibrium biased towards the T state in opossum iron-hemoglobin.     

EPR characterization of the stereochemistry of the distal heme pocket of the engineered human myoglobin mutants.

Recombinant human myoglobin mutants with the distal histidine residue replaced by Leu, Val, or Gln residues have been prepared by site-directed mutagenesis and expression in Escherichia coli. The recombinant apomyoglobin proteins have been successfully reconstituted with cobaltous protoporphyrin IX to obtain cobalt myoglobin mutant proteins, and the role of the distal histidine residue on the interaction between the bound ligand and the myoglobin molecule has been studied by EPR spectroscopy. We found that the distal histidine residue is significant in the orientation of the bound oxygen molecule. Low temperature photolysis experiments on both oxy cobalt proteins and ferric nitric oxide complexes indicated that the nature of the photolyzed form depends on the steric crowding of the distal heme pocket. To our surprise, the distal Leu mutant has a less restricted, less sterically crowded distal heme pocket than that of the distal Val mutant myoglobin, despite the fact that Leu has a larger side chain volume than Val. Our results demonstrate that the distal heme pocket steric crowding is not necessarily related to the side chain volume of the E7 residue.     

Hydrogen bonding to the bound dioxygen in oxy cobaltous myoglobin reduces the superhyperfine coupling to the proximal histidine.

Electron spin echo envelope modulation (ESEEM) spectroscopy was used to study the electron-nuclear coupling in two oxygenated cobalt-substituted hemoproteins, myoglobin (oxyCoMb) and a monomeric hemoglobin from Glycera dibranchiata (oxyCoHbgly). The modulation frequency components in ESEEM spectra of both proteins arose from the coupling to the N epsilon of the proximal histidyl imidazole. The hyperfine and quadrupole coupling parameters for these two nitrogens, calculated by computer spectral simulation, are Aiso = 2.46 MHz, e2qQ = 2.15 MHz, and eta = 0.4 for oxyCoMb and Aiso = 3.70 MHz, e2qQ = 2.70 MHz, and eta = 0.5 for oxyCoHbgly. A hyperfine coupling of 0.6 MHz, found for oxyCoMb in D2O but not for oxyCoHbgly in D2O, was assigned to the coupling to a deuteron that is hydrogen-bonded to the O2 ligand in oxyCoMb. This hydrogen bonding is believed to be responsible for the reduction in hyperfine and nuclear quadrupole coupling to the proximal histidyl imidazole N epsilon in oxyCoMb. A molecular orbital model for O2 adducts of cobaltous compounds [Tovrog et al. (1976) J. Am. Chem. Soc. 98, 5144] was used to understand the hydrogen bond-induced reduction in 14N superhyperfine coupling in oxyCoMb.     

The cobalt-nitrosyl stretching vibration as a sensitive resonance Raman probe for distal histidine-nitrosyl interaction in monomeric hemoglobins

The Co-NO stretching vibration has been assigned in the resonance Raman spectra of various cobalt-substituted monomeric hemoglobins by employing isotope-labeling of nitrosyl (14N16O, 15N16O, 14N18O). Monomeric hemoglobins with a distal histidine (sperm whale myoglobin and leghemoglobin) exhibit this vibration at 573-575 cm-1, whereas hemoglobins without distal histidine (elephant myoglobin and insect hemoglobin from Chironomus thummi thummi, CTT III) show this vibration in the range of 553-558 cm-1. The Fe-NO stretching vibration which occurs in the range of 554-556 cm-1 does not reflect the distal histidine-ligand interaction. Therefore, the Co-NO moiety which is isoelectronic with the Fe-O2 moiety is a good monitor for distal effects on the exogenous ligand of hemoglobins, especially due to the fact that in hemoglobins with distal histidine the Fe-O2 stretching vibration (567-572 cm-1) is similar to the Co-NO stretching vibration.     

pH Dependence of oxy and deoxy cobalt-substituted leghemoglobin from soybean

In leghemoglobin a, which is the major hemoglobin component in soybean root nodules, the haem iron has been replaced by cobalt. The electron spin resonance (ESR) of frozen solutions of the cobalt-substituted leghemoglobin has been studied at 77 K in the deoxy and oxy forms respectively. Both ligation states exhibit rhombic g tensors. The hyperfine constants of ⁵⁹Co, ¹⁴N-imidazole (residue of the proximal histidine) and ¹⁴N-pyrroles are determined for the three principal directions of the g tensor. Both, the oxy and the deoxy state exhibit pH-dependent changes of the hyperfine structures. For oxy cobalt leghemoglobin a quantitative analysis of the pH titration and of the ESR parameters of the low and high-pH forms respectively are performed. The interconversion of the low and the high-pH forms is controlled by a proton-dissociating group with pK=6.4 which is most probably the distal histidine. g tensors and hyperfine constants are compared with those described for oxy cobalt myoglobin crystal spectra [34] allowing assignments of the low and high-pH species of leghemoglobin to stereoelectronic structures with non-equivalent and equivalent dioxygen atoms respectively. Hydrogen-bonding of the distal histidine with dioxygen favours the structure with equivalent oxygen atoms. The pH dependence of the deoxy form is interpreted as interaction of the proximal imidazole with the central cobalt atom.     

Trans-substitution of the proximal hydrogen bond in myoglobin: II. Energetics, functional consequences, and implications for hemoglobin allostery

The trans-substituted histidine to glycine mutant of sperm whale myoglobin (H93G Mb) is used to study energetics of proximal hydrogen bonding, proximal ligand-heme interactions, and coupling to distal ligand binding. Comparison of mono- and dimethylimidazole structural isomers shows that the hydrogen bond between the proximal ligand and the neighboring Ser92 hydroxyl (position F7) is stabilizing. The range of hydrogen bond stabilities measured here for different distal ligand complexes ranges from -0.7 kcal/mol (monomethylimidazole isomers to MbCO) to -4.1 kcal/mol (dimethylimidazole isomers to MbCN). This range of hydrogen bond stabilities, which is similar to that seen in protein mutagenesis unfolding studies, demonstrates the high sensitivity of the hydrogen bond to modest structural perturbations. The degree to which the 2-methyl group destabilizes proximal ligand binding is found to depend inversely on the total electronic spin. For monomethylimidazole proximal ligands, distal ligand binding weakens the proximal hydrogen bond compared to deoxyMb. Surprisingly, this trend is largely reversed for the dimethylimidazole proximal ligands. These results demonstrate strong coupling between the proximal protein matrix and distal ligand binding. These results provide an explanation for the strong avoidance of hydrogen bonding residues at position F7 in hemoglobin sequences.     

Effect of the distal residues on the vibrational modes of the Fe-CO bond in hemoglobin studied by protein engineering

Using an Escherichia coli gene expression system, we have engineered human hemoglobin (Hb) mutants having the distal histidine (E7) and valine (E11) residues replaced by other amino acids. The interaction between the mutated distal residues and bound carbon monoxide has been studied by Soret-excited resonance Raman spectroscopy. The replacement of Val-E11 by Ala, Leu, Ile, and Met has no effect on the v(C-O), v(Fe-CO) stretching or delta(Fe-C-O) bending frequencies in both the alpha and beta subunits of Hb, although some of these mutations affect the CO affinity as much as 40-fold. The strain imposed on the protein by the binding of CO is not localized in the Fe-CO bond and is probably distributed among many bonds in the globin. The replacement of His-E7 by Val or Gly brings the stretching frequencies v(Fe-CO) and v(C-O) close to those of free heme complexes. In contrast, the substitution of His-E7 by Gln, which is flexible and polar, produces no effects on the resonance Raman spectrum of either alpha- or beta-globin. The replacement of His-E7 of beta-globin by Phe shows the same effect as replacement by Gly or Val. Therefore, the steric bulk of the distal residues is not the primary determinant of the Fe-CO ligand vibrational frequencies. The ability of both histidine and glutamine to alter the v(C-O), v(Fe-CO), or delta(Fe-C-O) frequencies may be attributed to the polar nature of their side chains which can interact with bound CO in a similar manner.     

Structural and electronic properties of the liver fluke heme cavity by nuclear magnetic resonance and optical spectroscopy. Evidence for a distal tyrosine residue in a normally functioning hemoglobin

Structural features of the heme and the heme cavity of the monomeric hemoglobin (Hb) from the platyhelminth Dicrocoelium dendriticum were investigated by optical and proton nuclear magnetic resonance spectroscopy. Using nuclear Overhauser effects (NOEs) from resonances assigned previously through isotope labeling, most hyperfine-shifted resonances could be attributed to individual heme and protein protons in the cyano-metHb complex. It was observed that the heme 2-vinyl group is held in the trans orientation by nearby residues, whereas the 4-vinyl group exhibits an equilibrium between cis and trans orientations. NOE experiments in 1H2O allowed the identification of exchangeable protons belonging to the proximal histidine residue (F8) and to a distal residue. Detailed analysis of the NOE patterns obtained from the distal labile proton to non-labile protons and among these latter protons leads to the conclusion that a tyrosine side-chain occupies the distal site E7. Optical spectra of the alkaline-metHb also lead to this view, in that they are not typical of a hydroxy-metHb complex but instead resemble that of a hemin-phenolate or human mutant (M-type) Hb with a tyrosine residue linked to the iron atom. Further evidence for a distal tyrosine residue stems from the occurrence of an unusually stable transient ferrous Hb-cyanide complex, formed upon reduction of cyano-metHb to deoxy-Hb with dithionite. We suggest that the stability of this intermediate is due to a slow re-orientation of a large distal side-chain prior to cyanide dissociation. The sequence of the E-helix, known from the partially determined primary structure, was realigned to accommodate these findings. A frame-shift by one residue now positions a tyrosine at the distal site E7 instead of the originally proposed glycine residue.     

High resolution NMR studies of histidine-substituted and histidine-perturbed hemoglobin variants. Histidine assignments, electrostatic interactions at the protein surface, and implications for hemoglobin S polymerization

The aromatic region of the proton NMR spectrum of human adult hemoglobin (HbA) contains resonances from at least 11 titratable histidine residues. Assignments for five beta chain histidines have previously been proposed. In order to further characterize the aromatic spectra of HbA we studied 11 histidine-substituted and -perturbed hemoglobin variants in oxy and deoxy states and at different pH values by 400 MHz NMR spectroscopy. We propose assignments for the resonances corresponding to the C2 protons of His alpha 20, His alpha 72, His alpha 112, and His beta 77 in oxy and deoxy spectra and of His beta 97 and His beta 117 in deoxy spectra. Our assignments for His beta 2 and His beta 117 in the oxy state agree with those previously reported for the CO form, but in the deoxy state our spectra suggest a different assignment. Studies with Hb variants in which a histidine is perturbed by a neighboring substitution suggest additional assignments for His alpha 50 and His alpha 89 and demonstrate a strong dependence of the imidazole ring pK on hydrogen bond interactions and on the net charge of neighboring residues. Some of the newly proposed assignments of histidine resonances are used to discuss specific intermolecular interactions implicating His alpha 20, His beta 77, and His beta 117 in deoxy HbS polymers.     

Unusual CO bonding geometry in abnormal subunits of hemoglobin M Boston and hemoglobin M Saskatoon

To clarify the role of the proximal histidine (F8-His), distal His (E7-His), and E11 valine (E11-Val) in ligand binding of hemoglobin (Hb), we have investigated the resonance Raman (RR) spectra of the carbon monoxide adduct of Hbs M (COHb M) in which one of these residues was genetically replaced by another amino acid in either the alpha or beta subunit. In the fully reduced state, all Hbs M gave v3 at approximately 1472 cm-1 and vFe-His at 214-218 cm-1, indicating that they have a pentacoordinate heme and the heme iron is bound to either E7-His or F8-His. The porphyrin skeletal vibrations of the COHb M were essentially unaltered by replacements of E7- or F8-His with tyrosine (Tyr) and of E11-Val by glutamic acid (Glu). The vCO, vFe-CO, and delta Fe-C-O frequencies of COHb M Iwate (alpha F8-His----Tyr), COHb M Hyde Park (beta F8-His----Tyr), and COHb M Milwaukee (beta E11-Val----Glu) were nearly identical with those of COHb A. In contrast, the RR spectra of COHb M Boston (alpha E7-His----Tyr) and COHb M Saskatoon (beta E7-His----Tyr) gave two new Raman bands derived from the abnormal subunits, vFe-CO at 490 cm-1 and vCO at 1972 cm-1, in addition to those from the normal subunits at 505 cm-1 (vFe-CO) and 1952 cm-1 (vCO). The CO adduct of the abnormal subunits exhibited apparently no photodissociation upon illumination of CW laser with a stationary cell under which the normal subunit exhibited complete photodissociation.(ABSTRACT TRUNCATED AT 250 WORDS)     

1H-NMR investigation of the oxygenation of hemoglobin in intact human red blood cells.

Using improved selective excitation methods for protein nuclear magnetic resonance (NMR), we have conducted measurements of the oxygenation of hemoglobin inside intact human red blood cells. The selective excitation methods use pulse shape-insensitive suppression of the water signal, while producing uniform phase excitation in the region of interest and, thus, are suitable for a wide variety of applications in vivo. We have measured the areas of 1H-NMR resonances of the hyperfine-shifted, exchangeable N delta H protons of the proximal histidine residues of the alpha- and beta-chains in deoxyhemoglobin (63 and 76 ppm downfield from the proton resonance of 2,2-dimethyl-2-silapentane-5-sulfonate (DSS), respectively), which are sensitive to the paramagnetic state of the iron, and for which the alpha- and beta-chain resonances are resolved, and from the ring current-shifted gamma 2-CH3 protons of the distal valine residues in oxyhemoglobin (2.4 ppm upfield from DSS), which are sensitive to the conformation of the heme pocket in the oxy state. We have found that the proximal histidine resonances are directly correlated with the degree of oxygenation of hemoglobin, whereas the distal valine resonances appear to be correlated with the conformation in the heme pocket that occurs after the binding of oxygen, in both the presence and absence of 2,3-diphosphoglycerate. In addition, from the proximal histidine resonances, we have observed a preference for the binding of oxygen to the alpha-chain (up to about 10%) of hemoglobin over the beta-chain in both the presence and absence of 2,3-diphosphoglycerate. These new results obtained in intact erythrocytes are consistent with our previous 1H-NMR studies on purified human normal adult hemoglobin. A unique feature of our 1H-NMR method is the ability to monitor the binding of oxygen specifically to the alpha- and beta-chains of hemoglobin both in solution and in intact red blood cells. This information is essential to our understanding of the molecular basis for the hemoglobin molecule serving as the oxygen carrier in vertebrates.     

The assignment of carbon monoxide association rate constants to the alpha and beta subunits in native and mutant human deoxyhemoglobin tetramers

The association kinetics of CO binding to site-directed mutants of human deoxyhemoglobin were measured by stopped-flow rapid mixing techniques at pH 7.0, 20 degrees C. Hemoglobin tetramers were constructed from one set of native subunits and one set of mutated partners containing His(E7) to Gly, Val(E11) to Ala, or Val(E11) to Ile substitutions. The reactivity of beta Cys93 with p-hydroxymercuribenzoate was measured to ensure that the mutant deoxyhemoglobins were capable of forming T-state quaternary conformations. Time courses for the complete binding of CO were measured by mixing the deoxygenated proteins with a 5-fold excess of ligand in the absence and presence of inositol hexaphosphate. Association rate constants for the individual alpha and beta subunits in the T-state conformation were assigned by measuring time courses for the reaction of a small, limiting amount of CO with a 20-fold excess of deoxyhemoglobin (i.e. Hb4 + CO----Hb4(CO)). The effects of the E7 and E11 mutations in T-state alpha subunits were qualitatively similar to those observed for the same subunit in the R-state (Mathews, A.J., Rohlfs, R.J., Olson, J.S., Tame, J., Renaud, J-P., and Nagai, K. (1989) J. Biol. Chem. 264, 16573-16583). The alpha His58(E7) to Gly and Val62(E11) to Ala substitutions caused 80- and 3-fold increases, respectively, in k'CO for T-state alpha subunits, and the alpha Val62(E11) to Ile mutation caused a 3-fold decrease. The beta His63(E7) to Gly and Val67(E11) to Ala substitutions produced 70- and 8-fold increases, respectively, in k'CO for T-state beta subunits whereas these same mutations caused little effect on the rate of CO binding to R-state beta subunits. The beta Val67(E11) to Ile mutation produced the same large effect, a 23-fold reduction in k'CO, in both quaternary conformations of beta subunits. These kinetic results can be interpreted qualitatively in terms of differences between the alpha and beta subunits in the deoxy and liganded crystal structures of human hemoglobin (Perutz, M.F. (1990) Annu. Rev. Physiol. 52, 1-25). Both the structural and functional data suggest that the distal portion of the beta heme pocket is tightly packed in deoxyhemoglobin whereas the CO binding site in R-state beta subunits is much more open. In contrast, the distal portion of the alpha heme pocket is restricted sterically in both quaternary states.(ABSTRACT TRUNCATED AT 400 WORDS)     

Engineering His(E7) affects the control of heme reactivity in Aplysia limacina myoglobin

Aplysia limacina myoglobin lacks the distal histidine (His (E7)) and displays a ligand stabilization mechanism based on Arg(E10). The double mutant Val(E7)His-Arg(E10)Thr has been prepared to engineer the role of His(E7), typical of mammalian myoglobins, in a different globin framework. The 2.0 A crystal structure of Val(E7)His-Arg(E10)Thr met-Mb mutant reveals that the His(E7) side chain points out of the distal pocket, providing an explanation for the observed failure to stabilize the Fe(II) bound oxygen in the ferrous myoglobin. Moreover, spectroscopic analysis together with kinetic data on azide binding to met-myoglobin are reported and discussed in terms of the presence of a water molecule at coordination distance from the heme iron.     

Hemoglobin tertiary structural change on ligand binding its role in the co-operative mechanism

Analysis of the tertiary structural alterations in hemoglobin induced by ligand binding demonstrates that an allosteric core composed of the heme, histidine F8, the FG corner and part of the F-helix plays an essential role in co-operativity. This conclusion is based on structural and spectroscopic data and theoretical studies of hemoglobin chains. The methodology employed in the calculations is presented with details of the empirical energy function. Energy minimized structures of the unliganded hemoglobin chains, which serve as reference systems for the analysis, are described. To determine the structural changes induced by ligand binding, the effects of FeN bond shortening and of heme translation and tilting perturbations are examined. Energy minimization in the presence of the perturbations serves to provide information concerning the globin structural modifications produced by them. The validity of the results is supported by comparisons with the X-ray data of Anderson, Pulsinelli, Baldwin and Chothia on tertiary changes in the hemoglobin subunits.Internal to the allosteric core, there appear to be two stable positions for its elements: one of these corresponds to the liganded and the other to the unliganded species. The unliganded geometry fits without strain into the deoxy tetramer, while the liganded one fits without strain into the oxy tetramer. On ligation of a subunit in the deoxy tetramer, the structural changes within the allosteric core are in the direction of those found in going from the unliganded deoxy to the liganded oxy system, although they are reduced by the presence of constraints due to the other subunits in the deoxy tetramer. In addition, the quaternary constraints in the deoxy tetramer prevent the large overall displacement of the allosteric core that occurs in the transition to the liganded oxy tetramer. The coupling between the changes internal to the allosteric core, produced on ligation and the overall displacement of the core that accompanies the quaternary transition, is an essential element of the co-operative mechanism. As shown in previous work (Gelin & Karplus, 1977), the proximal histidine serves as the link between the position of the heme and the F-helix; the asymmetric orientation of the histidine in the deoxy structure, coupled with contributions from other heme-protein interactions, appears to initiate the tertiary structural changes induced by ligand binding. The reduced oxygen affinity of hemoglobin results not from tension on the heme in the unliganded structure (there is none) but instead from strain in the liganded subunit of the tetramer within the deoxy quaternary structure. Further, the changes in the allosteric core provide a relatively localized reaction path for transmitting information concerning ligand binding from the heme group to the surface of the subunit; particularly in the α-chain, the residue Val FG5 appears to play an important role in the reaction path.The present analysis has important implications for realistic statistical thermodynamic models of hemoglobin co-operativity. It suggests that the previously formulated model (Szabo & Karplus, 1972) should be generalized by the introduction of two different subunit tertiary structures in the deoxy and in the oxy tetramer; they would be associated with the unliganded and the liganded allosteric core, respectively, and would take account of steric constraints that reduce the ligand affinity of the deoxy tetramer.     

Controlling ligand binding in myoglobin by mutagenesis.

A quadruple mutant of sperm whale myoglobin was constructed to mimic the structure found in Ascaris suum hemoglobin. The replacements include His(E7)-->Gln, Leu(B10)-->Tyr, Thr(E10)--> Arg, and Ile(G8)-->Phe. Single, double, and triple mutants were characterized to dissect out the effects of the individual substitutions. The crystal structures of the deoxy and oxy forms of the quadruple mutant were determined and compared with that of native Ascaris hemoglobin. Tyr(B10) myoglobin displays low O(2) affinity, high dissociation rate constants, and heterogeneous kinetic behavior, suggesting unfavorable steric interactions between the B10 phenol side chain and His(E7). In contrast, all mutants containing the Tyr(B10)/Gln(E7) pair show high O(2) affinity, low dissociation rate constants, and simple, monophasic kinetic behavior. Replacement of Ile(107) with Phe enhances nanosecond geminate recombination singly and in combination with the Tyr(B10)/Gln(E7)/Arg(E10) mutation by limiting access to the Xe4 site. These kinetic results and comparisons with native Ascaris hemoglobin demonstrate the importance of distal pocket cavities in governing the kinetics of ligand binding. The approximately 150-fold higher O(2) affinity of Ascaris hemoglobin compared with that for Tyr(B10)/Gln(E7)-containing myoglobin mutants appears to be the result of favorable proximal effects in the Ascaris protein, due to a staggered orientation of His(F8), the lack of a hydrogen bonding lattice between the F4, F7, and F8 residues, and the presence of a large polar Trp(G5) residue in the interior portion of the proximal heme pocket.     

The position 68(E11) side chain in myoglobin regulates ligand capture, bond formation with heme iron, and internal movement into the xenon cavities

After photodissociation, ligand rebinding to myoglobin exhibits complex kinetic patterns associated with multiple first-order geminate recombination processes occurring within the protein and a simpler bimolecular phase representing second-order ligand rebinding from the solvent. A smooth transition from cryogenic-like to solution phase properties can be obtained by using a combination of sol-gel encapsulation, addition of glycerol as a bathing medium, and temperature tuning (-15 --> 65 degrees C). This approach was applied to a series of double mutants, myoglobin CO (H64L/V68X, where X = Ala, Val, Leu, Asn, and Phe), which were designed to examine the contributions of the position 68(E11) side chain to the appearance and disappearance of internal rebinding phases in the absence of steric and polar interactions with the distal histidine. Based on the effects of viscosity, temperature, and the stereochemistry of the E11 side chain, the three major phases, B --> A, C --> A, and D --> A, can be assigned, respectively, to ligand rebinding from the following: (i) the distal heme pocket, (ii) the xenon cavities prior to large amplitude side chain conformational relaxation, and (iii) the xenon cavities after significant conformational relaxation of the position 68(E11) side chain. The relative amplitudes of the B --> A and C --> A phases depend markedly on the size and shape of the E11 side chain, which regulates sterically both ligand return to the heme iron atom and ligand migration to the xenon cavities. The internal xenon cavities provide a transient docking site that allows side chain relaxations and the entry of water into the vacated distal pocket, which in turn slows ligand recombination markedly.     

Proton nuclear magnetic resonance study of the solution distal histidine orientation in monomeric Chironomus thummi thummi cyanomet hemoglobins. Dynamic stability of the heme pocket as monitored by labile proton exchange.

The 1H nuclear magnetic resonance spectral characteristics of the cyano-Met form of Chironomus thummi thummi monomeric hemoglobins I, III and IV in 1H2O solvent are reported. A set of four exchangeable hyperfine-shifted resonances is found for each of the two heme-insertion isomers in the hyperfine-shifted region downfield of ten parts per million. An analysis of relaxation, exchange rates and nuclear Overhauser effects leads to assignments for all these resonances to histidine F8 and the side-chains of histidine E7 and arginine FG3. It is evident that in aqueous solution, the side-chain from histidine E7 does not occupy two orientations, as found for the solid state, rather the histidine E7 side-chain adopts a conformation similar to that of sperm whale myoglobin or hemoglobin A, oriented into the heme pocket and in contact with the bound ligand. Evidence is presented to show that the allosteric transition in the Chironomus thummi thummi hemoglobins arises from the "trans effect". An analysis of the exchange with bulk solvent of the assigned histidine E7 labile proton confirms that the group is completely buried within the heme pocket in a manner similar to that found for sperm whale cyano-Met myoglobin, and that the transient exposure to solvent is no more likely than in mammalian myoglobins with the "normal" distal histidine orientation. Finally, a comparison of solvent access to the heme pocket of the three monomeric C. thummi thummi hemoglobins, as measured from proton exchange rates of heme pocket protons, is made and correlated to binding studies with the diffusible small molecules such as O2.