Experimental evaluation of dual Multiple Aperture Devices for Fluence Field Modulated X-Ray Computed Tomography.

Acquisition of CT images with comparable diagnostic power can potentially be achieved with lower radiation exposure than the current standard of care through the adoption of hardware-based fluence-field modulation (e.g. dynamic bowtie filters). While modern CT scanners employ elements such as static bowtie filters and tube-current modulation, such solutions are limited in the fluence patterns that they can achieve, and thus are limited in their ability to adapt to broad classes of patient morphology. Fluence-field modulation also enables new applications such as region-of-interest imaging, task specific imaging, reducing measurement noise or improving image quality. The work presented in this paper leverages a novel fluence modulation strategy that uses "Multiple Aperture Devices" (MADs) which are, in essence, binary filters, blocking or passing x-rays on a fine scale. Utilizing two MAD devices in series provides the capability of generating a large number of fluence patterns via small relative motions between the MAD filters. We present the first experimental evaluation of fluence-field modulation using a dual-MAD system, and demonstrate the efficacy of this technique with a characterization of achievable fluence patterns and an investigation of experimental projection data.

Oligomerization and ligand binding in a homotetrameric hemoglobin: two high-resolution crystal structures of hemoglobin Bart's (gamma(4)), a marker for alpha-thalassemia.

Hemoglobin (Hb) Bart's is present in the red blood cells of millions of people worldwide who suffer from alpha-thalassemia. alpha-Thalassemia is a disease in which there is a deletion of one or more of the four alpha-chain genes, and excess gamma and beta chains spontaneously form homotetramers. The gamma(4) homotetrameric protein known as Hb Bart's is a stable species that exhibits neither a Bohr effect nor heme-heme cooperativity. Although Hb Bart's has a higher O(2) affinity than either adult (alpha(2)beta(2)) or fetal (alpha(2)gamma(2)) Hbs, it has a lower affinity for O(2) than HbH (beta(4)). To better understand the association and ligand binding properties of the gamma(4) tetramer, we have solved the structure of Hb Bart's in two different oxidation and ligation states. The crystal structure of ferrous carbonmonoxy (CO) Hb Bart's was determined by molecular replacement and refined at 1.7 A resolution (R = 21.1%, R(free) = 24.4%), and that of ferric azide (N(3)(-)) Hb Bart's was similarly determined at 1.86 A resolution (R = 18.4%, R(free) = 22.0%). In the carbonmonoxy-Hb structure, the CO ligand is bound at an angle of 140 degrees, and with an unusually long Fe-C bond of 2.25 A. This geometry is attributed to repulsion from the distal His63 at the low pH of crystallization (4.5). In contrast, azide is bound to the oxidized heme iron in the methemoglobin crystals at an angle of 112 degrees, in a perfect orientation to accept a hydrogen bond from His63. Compared to the three known quaternary structures of human Hb (T, R, and R2), both structures most closely resemble the R state. Comparisons with the structures of adult Hb and HbH explain the association and dissociation behaviour of Hb homotetramers relative to the heterotetrameric Hbs.

Haem disorder in recombinant- and reticulocyte-derived haemoglobins: evidence for stereoselective haem insertion in eukaryotes.

We have used NMR spectroscopy to measure haem disorder in adult human haemoglobin (HbA) obtained from mature erythrocyte cells and from yeast expressing recombinant HbA. Reticulocyte-derived HbA contained much higher levels of haem disorder (11% alpha- and 28% beta-subunit disorder) than observed for HbA from mature erythrocytes (1.5% alpha- and 8% beta-subunit disorder). Thus, unlike in vitro combination of haem and apoHb, biosynthetic haem insertion is not random with respect to orientation, but appears to show stereoselectivity. Recombinant HbA isolated from yeast showed 32% alpha- and 45% beta-subunit haem disorder. These levels relaxed to their equilibrium positions after incubating the Hb in the ferric form. Recombinant embryonic human Hbs showed less haem disorder than recombinant HbA. The levels of haem disorder in embryonic Hbs zeta(2)epsilon(2) and zeta(2)gamma(2) appear to have their equilibrium values. We propose that, in eukaryotes, in vivo haem insertion occurs via both co-translational mechanisms and insertion via semiHb-beta.

Application of high-precision isotope ratio monitoring mass spectrometry to identify the biosynthetic origins of proteins.

Isotope ratio monitoring (IRM) mass spectrometry was used to measure the relative abundance of stable isotopes in several samples of adult human hemoglobin expressed in E. coli, yeast, and human blood. The results showed significant differences in the distribution of (15)N and (13)C isotopes among hemoglobin samples produced in these organisms. This indicates that IRM mass spectrometry can be used in forensic protein chemistry to identify the origin of protein expression.

The use of subatmospheric pressure dressing for the coverage of radial forearm free flap donor-site exposed tendon complications.

Since its description in China in 1978, the radial forearm free flap has become a workhorse for the reconstructive surgeon. However, the flap has known disadvantages in complications of the wrist donor site. Skin graft breakdown with exposure of the flexor tendons of the wrist is the most common. The authors describe in a patient series a new treatment for this complication. They used subatmospheric pressure dressing to stimulate granulation tissue coverage of the tendon and to facilitate epithelialization. As many as one third of all patients undergoing radial forearm free flaps develop exposed tendon complications and may benefit from Vacuum Assisted Closure (VAC) therapy.

Acute and long-term stability studies of deoxy hemoglobin and characterization of ascorbate-induced modifications.

The reaction of ascorbate with recombinant hemoglobin (rHb1.1) in the presence of differing partial pressures of oxygen was studied. In the presence of 15 000 ppm (1.5%) residual oxygen, ascorbate/oxygen-mediated reactions resulted in an increased rate of autoxidation, modification of the beta-globin, increased oxygen affinity and decreased maximum Hill coefficient. One of the observed modifications to the beta-globin was a 72 Da addition to its N-terminus. Detailed characterization indicates the modification was an imidazolidinone type structure. Thorough deoxygenation of the hemoglobin solution to <150 ppm of oxygen prior to addition of ascorbate was required to prevent these modifications. Addition of ascorbate to the deoxy hemoglobin (deoxyHb) at pH 8 induced aggregation, eventually leading to precipitation. No such precipitation was observed at pH 7. Long-term storage of the hemoglobin was carried out by addition of ascorbate to deoxyHb at pH 7. The level of methemoglobin remained at <2% for up to 1 year at 4 degreesC, with no detectable precipitation of the protein. Modifications similar to those observed by the acute studies were observed over the 1-year period and correlated with disappearance of the added ascorbate.

Quaternary structure regulates hemin dissociation from human hemoglobin.

Rate constants for hemin dissociation from the alpha and beta subunits of native and recombinant human hemoglobins were measured as a function of protein concentration at pH 7.0, 37 degrees C, using H64Y/V68F apomyoglobin as a hemin acceptor reagent. Hemin dissociation rates were also measured for native isolated alpha and beta chains and for recombinant hemoglobin tetramers stabilized by alpha subunit fusion. The rate constant for hemin dissociation from beta subunits in native hemoglobin increases from 1.5 h-1 in tetramers at high protein concentration to 15 h-1 in dimers at low concentrations. The rate of hemin dissociation from alpha subunits in native hemoglobin is significantly smaller (0.3-0.6 h-1) and shows little dependence on protein concentration. Recombinant hemoglobins containing a fused di-alpha subunit remain tetrameric under all concentrations and show rates of hemin loss similar to those observed for wild-type and native hemoglobin at high protein concentration. Rates of hemin dissociation from monomeric alpha and beta chains are much greater, 12 and 40 h-1, respectively, at pH 7, 37 degrees C. Aggregation of monomers to form alpha1beta1 dimers greatly stabilizes bound hemin in alpha chains, decreasing its rate of hemin loss approximately 20-fold. In contrast, dimer formation has little stabilizing effect on hemin binding to beta subunits. A significant reduction in the rate of hemin loss from beta subunits does occur after formation of the alpha1beta2 interface in tetrameric hemoglobin. These results suggest that native human hemoglobin may have evolved to lose heme rapidly after red cell lysis, allowing the prosthetic group to be removed by serum albumin and apohemopexin.

Mechanism of NO-induced oxidation of myoglobin and hemoglobin.

Nitric oxide (NO) has been implicated as mediator in a variety of physiological functions, including neurotransmission, platelet aggregation, macrophage function, and vasodilation. The consumption of NO by extracellular hemoglobin and subsequent vasoconstriction have been suggested to be the cause of the mild hypertensive events reported during in vivo trials of hemoglobin-based O2 carriers. The depletion of NO from endothelial cells is most likely due to the oxidative reaction of NO with oxyhemoglobin in arterioles and surrounding tissue. In order to determine the mechanism of this key reaction, we have measured the kinetics of NO-induced oxidation of a variety of different recombinant sperm whale myoglobins (Mb) and human hemoglobins (Hb). The observed rates depend linearly on [NO] but show no dependence on [O2]. The bimolecular rate constants for NO-induced oxidation of MbO2 and HbO2 are large (k.ox,NO = 30-50 microM-1 s-1 for the wild-type proteins) and similar to those for simple nitric oxide binding to deoxygenated Mb and Hb. Both reversible NO binding and NO-induced oxidation occur in two steps: (1) bimolecular entry of nitric oxide into the distal portion of the heme pocket and (2) rapid reaction of noncovalently bound nitric oxide with the iron atom to produce Fe(2+)-N=O or with Fe(2+)-O-O delta- to produce Fe(3+)-OH2 and nitrate. Both the oxidation and binding rate constants for sperm whale Mb were increased when His(E7) was replaced by aliphatic residues. These mutants lack polar interactions in the distal pocket which normally hinder NO entry into the protein. Decreasing the volume of the distal pocket by replacing Leu(B10) and Val(E11) with aromatic amino acids markedly inhibits NO-induced oxidation of MbO2. The latter results provide a protein engineering strategy for reducing hypertensive events caused by extracellular hemoglobin-based O2 carriers. This approach has been explored by examining the effects of Phe(B10) and Phe(E11) substitutions on the rates of NO-induced oxidation of the alpha and beta subunits in recombinant human hemoglobin.

Differential scanning calorimetric study of the thermal unfolding transitions of yeast iso-1 and iso-2 cytochromes c and three composite isozymes.

The effects of regional sequence differences on the thermodynamic stability of a globular protein have been investigated by scanning microcalorimetry. Thermal transitions have been measured for two isozymes of yeast cytochrome c (iso-1-MS and iso-2) and three composite proteins (Comp1-MS, Comp2-MS, and Comp3-MS) in which amino acid segments are exchanged between the parental isozymes. There are three main observations. (1) In the temperature range of the unfolding transitions (40-60 degrees C) the unfolding free energies for the composite proteins are only slightly different from those of the parental isozymes, although in some cases there are large but compensating changes in the transitional enthalpy and entropy. At lower temperatures (0-30 degrees C), all the composites are significantly less stable than the two parental proteins. (2) Long-range structural effects are responsible for at least some of the observed differences in stability. For example, in the temperature range of the unfolding transitions (40-60 degrees C), the Comp1-MS protein which contains only a small amount of iso-2-like sequence is less stable than either of the parental isozymes, despite the fact that none of the iso-2-specific amino acid side chains impinges directly on any of the iso-1-specific amino acid side chains. (3) Changes in ionization of His 26 appear to be linked to thermal unfolding. Iso-1-MS and Comp1-MS contain a histidine residue at position 26 while iso-2 and the other two composites do not. On lowering the pH from pH 6 to 5, both iso-1-MS and Comp1-MS show a decrease in stability (lower Tm) within the unfolding transition region (40-60 degrees C), whereas the stabilities of iso-2, Comp2-MS, and Comp3-MS are essentially unchanged. The thermal unfolding transitions are highly reversible (> 95%) but mechanistically complex. A moderate dependence of Tm on protein concentration and the ratio of the van't Hoff enthalpy to the calorimetric enthalpy suggest that thermal unfolding involves the reversible association of a significant fraction of the unfolded species, at least at elevated protein concentrations.

His64(E7)-->Tyr apomyoglobin as a reagent for measuring rates of hemin dissociation.

To develop an assay for hemin dissociation, His64(E7) was replaced by Tyr in sperm whale myoglobin producing a holoprotein with a distinct green color due to an intense absorption band at 600 nm. Val68(E11) was replaced by Phe in the same protein to increase its stability. When excess Tyr64-Val68 apoglobin is mixed with either metmyoglobin or methemoglobin, the solution turns from brown to green, and the absorbance changes can be used to measure complete time courses for hemin dissociation from either holoprotein. This assay has been used to measure rates of hemin dissociation from native metmyoglobin, four myoglobin mutants (Ala64(E7), Ala68(E11), Phe68(E11), and Glu45(CD3)), native methemoglobin, valence hybrid hemoglobins, and two mutant hemoglobins ((alpha(Gly-E7)beta(native))2, and (alpha(native)beta(Gly-E7))2). Two kinetic phases were observed for hemin dissociation from native human hemoglobin at pH 7.0 and 37 degrees C. Valence and mutant hybrid hemoglobins were used to assign the faster phase (k = 7.8 +/- 2.0 h-1) to hemin dissociation from ferric beta subunits and the slower (k = 0.6 +/- 0.15 h-1) to dissociation from alpha subunits. The corresponding rate for wild-type metmyoglobin is 0.007 +/- 0.004 h-1.

A human recombinant haemoglobin designed for use as a blood substitute.

The need to develop a blood substitute is now urgent because of the increasing concern over blood-transmitted viral and bacterial pathogens. Cell-free haemoglobin solutions and human haemoglobin synthesized in Escherichia coli and Saccharomyces cerevisiae have been investigated as potential oxygen-carrying substitutes for red blood cells. But these haemoglobins cannot be used as a blood substitute because (1) the oxygen affinity in the absence of 2,3-bisphosphoglycerate is too high to allow unloading of enough oxygen in the tissues, and (2) they dissociate into alpha beta dimers that are cleared rapidly by renal filtration, which can result in long-term kidney damage. We have produced a human haemoglobin using an expression vector containing one gene encoding a mutant beta-globin with decreased oxygen affinity and one duplicated, tandemly fused alpha-globin gene. Fusion of the two alpha-globin subunits increases the half-life of this haemoglobin molecule in vivo by preventing its dissociation into alpha beta dimers and therefore also eliminates renal toxicity.

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)

Some reactions of carbon monoxide and oxygen with carbodi-imide-modified cytochrome c.

The reactivity of carbodi-imide-modified tuna and horse heart cytochromes c with the ferrous ion ligands CO and O2 has been studied. Both modified cytochromes bind one molecule of CO. Stopped-flow and flash-photolysis experiments indicate the presence of three kinetic processes in the reaction of the cytochromes with CO. The second-order rate constants associated with all three kinetic process are pH-independent being 2.8 x 10(5) M-1.s-1, 3.8 x 10(4) M-1.s-1 and 4 x 10(3) M-1.s-1 under all conditions studied. The concentration-dependence of the contributions made by each of the processes to the overall absorbance change indicates that the fast and slow kinetic phases are associated with two forms of the cytochromes which are in equilibrium, whereas the intermediate phase arises from a separate cytochrome species. The quantum yield for the photodissociation of CO from the ferrous cytochromes is unusually low. Both modified cytochromes are capable of binding and reducing O2. In the presence of excess reductant, the modified cytochromes can catalytically reduce large molar excesses of O2. In the absence of excess reducing agent, the oxy complex initially formed undergoes a pH-dependent intramolecular electron-transfer process with half-life approx. 10 min. EDC [1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide]-promoted internal cross-linking is proposed to account for differences between the EDC-modified proteins and carboxymethylated cytochrome c.

Differential stability of two apo-isocytochromes c in the yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae contains two forms of cytochrome c, iso-1-cytochrome c and iso-2-cytochrome c, encoded by the genes CYC1 and CYC7, respectively. The amino acid sequences of these two isozymes are approximately 80% identical. Cyc3- mutants lack both holocytochromes c, because of a deficiency of cytochrome c heme lyase, the enzyme catalyzing covalent attachment of the heme group to apocytochrome c. A deficiency of heme lyase also prevents import into mitochondria. Surprisingly, apo-iso-1-cytochrome c is absent in cyc3- strains, although apo-iso-2-cytochrome c is present at approximately the same level at which holo-iso-2-cytochrome c is found in CYC3+ strains. The lack of apo-iso-1-cytochrome c is not due to a deficiency of either transcription or translation, but to rapid degradation of the protein. Apocytochromes c encoded by composite cytochrome c genes composed of the central portion of iso-2-cytochrome c flanked by amino and carboxyl regions of iso-1-cytochrome c exhibit increased stability compared with apo-iso-1-cytochrome c. A region encompassing no more than four amino acid differences between iso-1- and iso-2-cytochromes c is sufficient to partially stabilize the protein. In contrast to what is observed in vivo with the apo forms, the holo forms of the composite isocytochromes c are even less stable to thermal denaturation than iso-1-cytochrome c or iso-2-cytochrome c. Either a small region of the sequence of apo-iso-1-cytochrome c is involved in degradation of the protein, or the corresponding region in apo-iso-2-cytochrome c is preventing degradation. The differential stability of the two isocytochromes c may be part of a regulatory process that increases the proportion of iso-2-cytochrome c under certain physiological conditions.

The effects of amino acid substitution at position E7 (residue 64) on the kinetics of ligand binding to sperm whale myoglobin.

Association and dissociation rate constants were measured for O2, CO, and alkyl isocyanide binding to a set of genetically engineered sperm whale myoglobins with site-specific mutations at residue 64 (the E7 helical position). Native His was replaced by Gly, Val, Leu, Met, Phe, Gln, Arg, and Asp using the synthetic gene and expression system developed by Springer and Sligar (Springer, B. A., and Sligar, S. G. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8961-8965). The His64----Gly substitution produced a sterically unhindered myoglobin that exhibited ligand binding parameters similar to those of chelated protoheme suspended in soap micelles. The order of the association rate constants for isocyanide binding to the mutant myoglobins was Gly64 (approximately 10(7) M-1 s-1) much greater than Val64 approximately Leu64 (approximately 10(6) M-1 s-1) greater than Met64 greater than Phe64 approximately His64 approximately Gln64 (10(5)-10(3) M-1 s-1) and indicates that the barrier to isocyanide entry into the distal pocket is primarily steric in nature. The bimolecular rates of methyl, ethyl, n-propyl, and n-butyl isocyanide binding to the His64----Arg and His64----Asp mutants were abnormally high (1-5 x 10(6) M-1 s-1), suggesting that Arg64 and Asp64 adopt conformations with the charged side chains pointing out toward the solvent creating a less hindered pathway for ligand binding. In contrast to the isocyanide data, the association rate constants for O2 and CO binding exhibited little dependence on the size of the E7 side chain. The values for all the mutants except His64----Gln approached or were larger than those for chelated model heme (i.e. approximately 1 x 10(8) M-1 s-1 for O2 and approximately 1 x 10(7) M-1 s-1 for CO), whereas the corresponding rate parameters for myoglobin containing either Gln64 or His64 were 5- to 10-fold smaller. This result suggests that a major kinetic barrier for O2 and CO binding to native myoglobin may involve disruption of polar interactions between His64 and water molecules found in the distal pocket of deoxymyoglobin. Finally, the rate and equilibrium parameters for O2 and CO binding to the His64----Gln, His64----Val, and His64----Leu mutants were compared to those reported previously for Asian elephant myoglobin (Gln-E7), Aplysia limacina myoglobin (Val-E7), and monomeric Hb II from Glycera dibranchiata (Leu-E7).

The effects of E7 and E11 mutations on the kinetics of ligand binding to R state human hemoglobin.

Association and dissociation rate constants for O2, CO, and methyl isocyanide binding to native and distal pocket mutants of R state human hemoglobin were measured using ligand displacement and partial photolysis techniques. Individual rate constants for the alpha and beta subunits were resolved by comparisons between the kinetic behavior of the native and mutant proteins. His-E7 was replaced with Gly and Gln in both alpha and beta subunits and with Phe in beta subunits alone. In separate experiments Val-E11 was replaced with Ala, Leu, and Ile in each globin chain. The parameters describing ligand binding to R state alpha subunits are sensitive to the size and polarity of the amino acids at positions E7 and E11. The distal histidine in this subunit inhibits the bimolecular rate of binding of both O2 and CO, sterically hinders bound CO and methyl isocyanide, and stabilizes bound O2 by hydrogen bonding. The Val-E11 side chain in alpha chains also appears to be part of the kinetic barrier to O2 and CO binding since substitution with Ala causes approximately 10-fold increases in the association rate constants for the binding of these diatomic ligands. However, substitution of Val-E11 by Ile produces only small decreases in the rates of ligand binding to alpha subunits. For R state beta subunits, the bimolecular rates of O2 and CO binding are intrinsically large, approximately 2-5-fold greater than those for alpha subunits, and with the exception of Val-E11----Ile mutation, little affected by substitutions at either the E7 or E11 positions. For the beta Val-E11----Ile mutant the association rate and equilibrium constants for all three ligands decreased 10-50-fold. All of these results agree with Shaanan's conclusions that the distal pocket in liganded beta subunits is more open whereas in alpha subunits bound ligands are more sterically hindered by adjacent distal residues (Shaanan, B. (1983) J. Mol. Biol. 171, 31-59). In the case of O2 binding to alpha subunits, the unfavorable steric effects are compensated by the formation of a hydrogen bond between the nitrogen atom of His-E7 and bound dioxygen.

Discrimination between oxygen and carbon monoxide and inhibition of autooxidation by myoglobin. Site-directed mutagenesis of the distal histidine.

Sperm whale myoglobin mutants were constructed using site-directed mutagenesis to replace the highly conserved distal histidine residue (His(E7)-64). His-64 was substituted with Gly, Val, Phe, Cys, Met, Lys, Arg, Asp, Thr, and Tyr, and all 10 mutant proteins expressed to approximately 10% of the total soluble cell protein in Escherichia coli as heme containing myoglobin. With the exception of His-64----Tyr, which did not form a stable oxygen (O2) complex, all mutant proteins could be reduced and bound O2 and carbon monoxide (CO) reversibly. However, removal of the distal histidine increased the rate of autooxidation 40-350-fold. The His-64----Gly, Val, Phe, Met, and Arg mutants all showed markedly increased O2 dissociation rate constants which were approximately 50-1500-fold higher than those for wild-type myoglobin and increased O2 association rate constants which were approximately 5-15-fold higher than those for the native protein. All mutants studied (except His-64----Tyr) showed approximately 10-fold increased CO association rates and relatively unchanged CO dissociation rates. These altered O2 and CO association and dissociation rate constants resulted in 3-14-fold increased CO affinities, 10-200-fold decreased O2 affinities, and 50-380-fold greater M (KCO/KO2) values for the mutants compared to the wild-type protein. Thus, the distal histidine of myoglobin discriminates between CO and O2 binding by both sterically hindering bound CO and stabilizing bound O2 through hydrogen bonding. The increased autooxidation rates observed for the mutants appear to be due to a decrease in oxygen affinity and an increase in solvent anion accessibility to the distal pocket.

The role of the distal histidine in myoglobin and haemoglobin.

The distal E7 histidine in vertebrate myoglobins and haemoglobins has been strongly conserved during evolution and is thought to be important in fine-tuning the ligand affinities of these proteins. A hydrogen bond between the N epsilon proton of the distal histidine and the second oxygen atom may stabilize O2 bound to the haem iron. The proximity of the imidazole side chain to the sixth coordination position, which is required for efficient hydrogen bonding, has been postulated to inhibit sterically the binding of CO and alkyl isocyanides. To test these ideas, engineered mutants of sperm whale myoglobin and the alpha- and beta-subunits of human haemoglobin were prepared in which E7 histidine was replaced by glycine. Removal of the distal imidazole in myoglobin and the alpha-subunits of intact, R-state haemoglobin caused significant changes in the affinity for oxygen, carbon monoxide and methyl isocyanide; in contrast, the His-E7 to Gly substitution produced little or no effect on the rates and extents of O2, CO and methyl isocyanide binding to beta-chains within R-state haemoglobin. In the beta-subunit the distal histidine seems to be less significant in regulating the binding of ligands to the haem iron in the high affinity quaternary conformation. Structural differences in the oxygen binding pockets shown by X-ray crystallographic studies account for the functional differences of these proteins.