Inversion of axial coordination in myoglobin to create a "proximal" ligand binding pocket

A ligand binding pocket has been created on the proximal side of the heme in porcine myoglobin by site-directed mutagenesis. Our starting point was the H64V/V68H double mutant which has been shown to have bis-histidine (His68 and His93) heme coordination [Dou, Y., Admiraal, S. J., Ikeda-Saito, M., Krzywda, S., Wilkinson, A. J., Li, T., Olson, J. S., Prince, R. C., Pickering, I. J., George, G. N. (1995) J. Biol. Chem. 270, 15993-16001]. The replacement of the proximal His93 ligand by noncoordinating Ala (H64V/V68H/H93A) or Gly (H64V/V68H/H93G) residues resulted unexpectedly in a six-coordinate low-spin species in both ferric and ferrous states. To test the hypothesis that the sixth coordinating ligand in the triple mutants was the imidazole of His97, this residue was mutated to Phe, in the quadruple mutants, H64V/V68H/H93A/H97F and H64V/V68H/H93G/H97F. The ferric quadruple mutants show a clear water/hydroxide alkaline transition and high cyanide and CO affinities, characteristics similar to those of wild-type myoglobin. The nu(Fe-CO) and nu(C-O) stretching frequencies in the ferrous-CO state of the quadruple mutants indicate that the "proximal" ligand binding heme pocket is less polar than the distal pocket in the wild-type protein. Thus, we conclude that the proximal heme pocket in the quadruple mutants has a similar affinity for exogenous ligands to the distal pocket of wild-type myoglobin but that the two pockets have different polarities. The quadruple mutants open up new approaches for developing heme chemistry on the myoglobin scaffold.     

Viscosity-dependent relaxation significantly modulates the kinetics of CO recombination in the truncated hemoglobin TrHbN from Mycobacterium tuberculosis

Kinetic traces were generated for the nanosecond and slower rebinding of photodissociated CO to trHbN in solution and in porous sol-gel matrices as a function of viscosity, conformation, and mutation. TrHbN is one of the two truncated hemoglobins from Mycobacterium tuberculosis. The kinetic traces were analyzed in terms of three distinct phases. These three phases are ascribed to rebinding: (i) from the distal heme pocket, (ii) from the adjacent apolar tunnel prior to conformational relaxation, and (iii) from the apolar tunnel subsequent to conformational relaxation. The fractional content of each of these phases was shown to be a function of the viscosity and, in the case of the sol-gel-encapsulated samples, sample preparation history. The observed kinetic patterns support a model consisting of the following elements: (i) the viscosity and conformation-sensitive dynamics of the Tyr(B10) side chain facilitate diffusion of the dissociated ligand from the distal heme pocket into the adjacent tunnel; (ii) the distal heme pocket architecture determines ligand access from the tunnel back to the heme iron; (iii) the distal heme pocket architecture is governed by a ligand-dependent hydrogen bonding network that limits the range of accessible side chain positions; and (iv) the apolar tunnel linking the heme site to the solvent biases the competition between water and ligand for occupancy of the vacated polar distal heme pocket greatly toward the nonpolar ligand. Implications of these finding with respect to biological function are discussed.     

Heme pocket disorder in myoglobin: reversal by acid-induced soft refolding

The protein folding process of heme proteins entails generation of not only a correct global polypeptide structure, but also a correct, functionally competent heme environment. We employed a variety of spectroscopic approaches to probe the structure and dynamics of the heme pocket of a recombinant sperm whale myoglobin. The conformational characteristics were examined by circular dichroism, time-resolved fluorescence spectroscopy, FTIR spectroscopy, and optical absorption spectroscopy in the temperature range 300-20 K. Each of these spectroscopic probes detected modifications confined exclusively to the heme pocket of the expressed myoglobin relative to the native protein. The functional properties were examined by measuring the kinetics of CO binding after flash-photolysis. The kinetics of the expressed myoglobin were more heterogeneous than those of the native protein. Mild acid exposure of the ferric derivative of the recombinant protein resulted in a protein with "nativelike" spectroscopic properties and homogeneous CO binding kinetics. The heme pocket modifications observed in this recombinant myoglobin do not derive from inverted heme. In contrast, when native apomyoglobin is reconstituted with the heme in vitro, the heme pocket disorder could be attributed exclusively to 180 degrees rotation of the bound heme [La Mar, G. N., Toi, H., and Krishnamoorthi, R. (1984) J. Am. Chem. Soc. 106, 6395-6401; Light, W. R., Rohlfs, R. J., Palmer, G., and Olson, J. S. (1987) J. Biol. Chem. 262, 46-52]. We conclude that exposure to low pH decreases the affinity of globin for the heme and allows an extended conformational sampling or "soft refolding" to a nativelike conformation.     

CO recombination in cytochrome c peroxidase: effect of the local heme environment on CO binding explored through site-directed mutagenesis

CO recombination to the cloned cytochrome c peroxidase [CCP(MI)] and mutants of CCP(MI) prepared by site-directed mutagenesis was examined as a function of pH by flash photolysis. The mutants examined included distal Arg 48----Leu, Lys; proximal Asp 235----Asn; and His 181----Gly. At alkaline pH, ferrous CCP(MI) was converted to a hexacoordinate form by a cooperative two-proton ionization, apparent pK(a) = 8.0. This change was observed in all of the mutants, although in the His 181----Gly mutant, the conversion to the hexacoordinate form was the result of a single-proton ionization, implicating His 181 as one of the two residues deprotonated in this isomerization. The pH-dependent conversion of CO ferrous CCP(MI) from acidic to alkaline forms was also observed and was similar to that reported for cytochrome c peroxidase from bakers' yeast [Iizuka, T., Makino, R., Ishimura, Y., & Yonetani, T. (1985) J. Biol. Chem. 260, 1407-1412]. Photolysis of the acidic form of the CO complex of CCP(MI) produces a kinetic form of the ferrous enzyme (form A) which exhibits the slow rate of CO recombination (l1' approximately 10(3) M-1 s-1) characteristic of peroxidases, while photolysis of the alkaline form of the CO complex produces a second kinetic form (form B), which exhibits a much faster rate of recombination (l2' approximately 10(5) M-1 s-1). Kinetic forms analogous to forms A and B were observed in all of the mutants examined. A third kinetic form (form B*) with a bimolecular rate constant l3' approximately 10(6) M-1 s-1 was also observed in the mutants at alkaline pH. Although the pH dependence for the conversion of form A to form B with increasing pH was altered by changes in the local heme environment, the rate of CO recombination by the respective forms was not dramatically altered in the mutants. Transient spectra of the reaction of CO with ferrous CCP(MI) after photolysis show that equilibrium between penta- and hexacoordinate ferrous enzyme is rapid relative to CO recombination. The presence of the internal sixth ligand has no discernible effect on the observed rate of recombination, however. The results presented indicate that in CCP(MI) the rate of ligand binding is determined primarily by isomerization of the protein from a closed conformation at acidic pH to an open conformation at alkaline pH and that polar effects of proximal Asp 235 and distal Arg 48 are of minor significance in the rate of CO recombination in both conformations.     

Ligand selectivity of soluble guanylyl cyclase: effect of the hydrogen-bonding tyrosine in the distal heme pocket on binding of oxygen, nitric oxide, and carbon monoxide

Although soluble guanylyl cyclase (sGC) functions in an environment in which O(2), NO, and CO are potential ligands for its heme moiety, the enzyme displays a high affinity for only its physiological ligand, NO, but has a limited affinity for CO and no affinity for O(2). Recent studies of a truncated version of the sGC beta(1)-subunit containing the heme-binding domain (Boon, E. M., Huang, S H., and Marletta, M. A. (2005) Nat. Chem. Biol., 1, 53-59) showed that introduction of the hydrogen-bonding tyrosine into the distal heme pocket changes the ligand specificity of the heme moiety and results in an oxygen-binding sGC. The hypothesis that the absence of hydrogen-bonding residues in the distal heme pocket is sufficient to provide oxygen discrimination by sGC was put forward. We tested this hypothesis in a context of a complete sGC heterodimer containing both the intact alpha(1)- and beta(1)-subunits. We found that the I145Y substitution in the full-length beta-subunit of the sGC heterodimer did not produce an oxygen-binding enzyme. However, this substitution impeded the association of NO and destabilized the NO.heme complex. The tyrosine in the distal heme pocket also impeded both the binding and dissociation of the CO ligand. We propose that the mechanism of oxygen exclusion by sGC not only involves the lack of hydrogen bonding in the distal heme pocket, but also depends on structural elements from other domains of sGC.     

A comparison of the geminate recombination kinetics of several monomeric heme proteins

The geminate rate constants for CO, O2, NO, methyl, ethyl, n-propyl, and n-butyl isocyanide rebinding to soybean leghemoglobin and monomeric component II of Glycera dibranchiata hemoglobin were measured at pH 7, 20 degrees C using a dye laser with a 30-ns square-wave pulse. The results were compared to the corresponding parameters for sperm whale myoglobin and the isolated alpha and beta subunits of human hemoglobin (Olson, J.S., Rohlfs, R.J., and Gibson, Q.H. (1987) J. Biol. Chem., 262, 12930-12938). The rate-limiting step for O2, NO, and isonitrile binding to all five proteins is ligand migration up to the initial geminate state, and the rate of this process determines the overall bimolecular association rate constant for these ligands. In contrast, iron-ligand bond formation limits the overall bimolecular rate for CO binding. The distal pockets in leghemoglobin and in Glycera HbII are approximately 10 times more accessible kinetically to diatomic ligands than that in sperm whale myoglobin. This difference accounts for the much larger association rate constants (1-2 x 10(8) M-1 s-1) that are observed for O2 and NO binding to leghemoglobin and Glycera HbII. The rates of isonitrile migration through leghemoglobin are also very large and indicate a very fluid or open distal structure near the sixth coordination position. In contrast, there is a marked decrease in the rate of migration up to and away from the sixth coordination position in Glycera HbII with increasing ligand size. These results were also used to interpret previously published rate constants and quantum yields for the high (R) and low (T) affinity states of human hemoglobin. In contrast to the differences between the monomeric proteins, the differences between the CO-, O2-, and NO-binding parameters for R and T state hemoglobin appear to be due to a decrease in the geminate reactivity of the heme iron atom, with little or no change in the accessibility of the distal pocket.     

X-ray structure and refinement of carbon-monoxy (Fe II)-myoglobin at 1.5 A resolution

The structure of carbon-monoxy (Fe II) myoglobin at 260 K has been solved at a resolution of 1.5 A by X-ray diffraction and a model refined against the X-ray data by restrained least-squares. The CO ligand is disordered and distorted from the linear conformation seen in model compounds. At least two conformations, with Fe--C--O angles of 140 degrees and 120 degrees, are required to model the system. The heme pocket is significantly larger than in deoxy-myoglobin because the distal residues have relaxed around the ligand; the largest displacement occurs for the distal histidine side-chain, which moves more than 1.4 A on ligand binding. The side-chain of Arg45 (CD3) is disordered and apparently exists in two equally populated conformations. One of these does not block the motion of the distal histidine out of the binding pocket, suggesting a mechanism for ligand entry. The heme group is planar (root-mean-square deviation from planarity is 0.08 A) with no doming of the pyrrole groups. The Fe--N epsilon 2 (His93) bond length is 2.2 A and the Fe--C bond length in the CO complex is 1.9 A. The iron is the least-squares plane of the heme, and this leads to the proximal histidine moving by 0.4 A relative to its position in deoxy-myoglobin. This shift correlates with a global structural change, with the proximal part of the molecule translated towards the heme plane.     

Structure of the yeast cytochrome bc1 complex with a hydroxyquinone anion Qo site inhibitor bound

Bifurcated electron transfer during ubiquinol oxidation is the key reaction of cytochrome bc1 complex catalysis. Binding of the competitive inhibitor 5-n-heptyl-6-hydroxy-4,7-dioxobenzothiazole to the Qo site of the cytochrome bc1 complex from Saccharomyces cerevisiae was analyzed by x-ray crystallography. This alkylhydroxydioxobenzothiazole is bound in its ionized form as evident from the crystal structure and confirmed by spectroscopic analysis, consistent with a measured pKa = 6.1 of the hydroxy group in detergent micelles. Stabilizing forces for the hydroxyquinone anion inhibitor include a polarized hydrogen bond to the iron-sulfur cluster ligand His181 and on-edge interactions via weak hydrogen bonds with cytochrome b residue Tyr279. The hydroxy group of the latter contributes to stabilization of the Rieske protein in the b-position by donating a hydrogen bond. The reported pH dependence of inhibition with lower efficacy at alkaline pH is attributed to the protonation state of His181 with a pKa of 7.5. Glu272, a proposed primary ligand and proton acceptor of ubiquinol, is not bound to the carbonyl group of the hydroxydioxobenzothiazole ring but is rotated out of the binding pocket toward the heme bL propionate A, to which it is hydrogen-bonded via a single water molecule. The observed hydrogen bonding pattern provides experimental evidence for the previously proposed proton exit pathway involving the heme propionate and a chain of water molecules. Binding of the alkyl-6-hydroxy-4,7-dioxobenzothiazole is discussed as resembling an intermediate step of ubiquinol oxidation, supporting a single occupancy model at the Qo site.     

Ligand binding and hexacoordination in synechocystis hemoglobin.

A large and phylogenetically diverse group of organisms contain truncated hemoglobins, including the unicellular cyanobacterium Synechocystis (Pesce, A., Couture, M., Dewilde, S., Guertin, M., Yamauchi, K., Ascenzi, P., Moens, L., and Bolognesi, M. (2000) EMBO J. 19, 2424-2434). Synechocystis hemoglobin is also hexacoordinate, with a heme pocket histidine that reversibly coordinates the ligand binding site. Hexacoordinate hemoglobins are ubiquitous in plants and are now being identified in a diverse array of organisms including humans (Arredondo-Peter, R., Hargrove, M. S., Moran, J. F., Sarath, G., and Klucas, R. V. (1998) Plant Physiol. 118, 1121-1125; Trent, J. T., III, Watts, R. A., and Hargrove, M. S. (2001) J. Biol. Chem. 276, 30106-30110). Rate constants for association and dissociation of the hexacoordinating amino acid side chain in Synechocystis hemoglobin have been measured along with bimolecular rate constants for association of oxygen and carbon monoxide following laser flash photolysis. These values were compared with ligand binding initiated by rapid mixing. Site-directed mutagenesis was used to determine the roles of several heme pocket amino acids in facilitating hexacoordination and stabilizing bound oxygen. It is demonstrated that Synechocystis hemoglobin contains a very reactive binding site and that ligand migration through the protein is rapid. Rate constants for hexacoordination by His(46) are also large and facilitated by other heme pocket amino acids including Gln(43).     

Water penetration and binding to ferric myoglobin

Flash photolysis investigations of horse heart metmyoglobin bound with NO (Mb(3+)NO) reveal the kinetics of water entry and binding to the heme iron. Photodissociation of NO leaves the sample in the dehydrated Mb(3+) (5-coordinate) state. After NO photolysis and escape, a water molecule enters the heme pocket and binds to the heme iron, forming the 6-coordinate aquometMb state (Mb(3+)H2O). At longer times, NO displaces the H2O ligand to reestablish equilibrium. At 293 K, we determine a value k(w) approximately 5.7 x 10(6) s(-1) for the rate of H2O binding and estimate the H2O dissociation constant as 60 mM. The Arrhenius barrier height H(w) = 42 +/- 3 kJ/mol determined for H2O binding is identical to the barrier for CO escape after photolysis of Mb(2+)CO, within experimental uncertainty, consistent with a common mechanism for entry and exit of small molecules from the heme pocket. We propose that both processes are gated by displacement of His-64 from the heme pocket. We also observe that the bimolecular NO rebinding rate is enhanced by 3 orders of magnitude both for the H64L mutant, which does not bind water, and for the H64G mutant, where the bound water is no longer stabilized by hydrogen bonding with His-64. These results emphasize the importance of the hydrogen bond in stabilizing H2O binding and thus preventing NO scavenging by ferric heme proteins at physiological NO concentrations.     

Resonance Raman evidence that distal histidine protonation removes the steric hindrance to upright binding of carbon monoxide by myoglobin

The resonance Raman band assigned to Fe--CO stretching in the sperm whale myoglobin CO adduct shifts from 507 cm-1 at neutral pH to 488 cm-1 at low pH, in concert with a shift of the C-O stretching infrared band from 1947 to 1967 cm-1 (Fuchsman & Appleby, 1979), while the 575-cm-1 Fe-C-O bending RR band loses intensity. The pKa that characterizes these changes is approximately 4.4. The vibrational frequencies at low pH are well modeled by the protein-free CO, imidazole adduct of protoheme in a nonpolar solvent while those at high pH are modeled by the adduct of a heme with a covalent strap (Yu et al., 1983) which inhibits upright CO binding. It is inferred that the Fe-C-O unit changes from a tilted to an upright geometry when the distal histidine is protonated, because its side chain swings out of the heme pocket due to electrostatic repulsion with a nearby arginine residue. A different protonation step (pKa = 5.7), which has been shown to modulate the CO rebinding kinetics (Doster et al., 1982) as well as the optical spectrum (Fuchsman & Appleby, 1979), is suggested to involve a global structure change associated with protonation of histidine residues distant from the heme.     

Ligand binding subsequent to CO photolysis of methionine-modified cytochrome c

In this report the kinetics of CO recombination to ferrocytochrome c in which Met80 has been oxidized to a sulfoxide are examined. Transient optical difference spectra suggest that the species formed immediately after photolysis contains a five-coordinate high spin heme. Single wavelength transient absorption data show triphasic kinetics with rate constants of (2.1+/-0.08)x10(4), (2.0+/-0.01)x10(3), and 57+/-0.01 s(-1). The data suggest a model for ligand recombination in which the methionine sulfoxide and CO compete for binding to the five-coordinate heme with rate constants of (2.1+/-0.08)x10(4) and (2.0+/-0.01)x10(3) s(-1), respectively. Carbon monoxide then binds to the population of cytochrome c containing the methionine sulfoxide with a rate constant of 57 s(-1). In addition, the slower than expected rate of methionine sulfoxide recombination (much smaller rate constant than expected for a ligand restricted to the distal heme pocket) is attributed to hydrogen bonding between the unbound methionine sulfoxide and Tyr(68).     

Ligand recombination to the alpha and beta subunits of human hemoglobin

The rebinding of CO, O2, NO, methyl, ethyl, n-propyl, and n-butyl isocyanide to isolated alpha and beta chains and intact hemoglobin at pH 7, 20 degrees C was examined both during and after a 30-ns dye laser pulse. The resultant absorbance changes were analyzed in terms of a linear three-step reaction scheme: Hb + X in equilibrium with C in equilibrium with B in equilibrium with A or HbX, where A is the final bound state, and C and B are geminate states. Rate constants were assigned for each of the transitions in this mechanism using fitting procedures described previously for analyzing ligand rebinding to sperm whale myoglobin at room temperature (Gibson, Q. H., Olson, J. S., McKinnie, R. E., and Rohlfs, R. J. (1986) J. Biol. Chem. 261, 10228-10239). Five major conclusions were obtained. First, initial geminate recombination phases for the NO and O2 complexes of hemoglobin and its isolated subunits exhibit half-times equal to approximately 12 and approximately 440 ps, respectively. These values are in excellent agreement with more direct, picosecond measurements of the geminate recombination of HbNO (Cornelius, P. A., Hochstrasser, R. M., and Steele, A. W. (1983) J. Mol. Biol. 163, 119-128) and HbO2 (Friedman, J. M., Scott, T. W., Fisanick, G. J., Simon, S. R., Findsen, E. W., Ondrias, M. R., and MacDonald, V. W. (1985) Science 229, 187-229) following extremely short laser pulses. Second, the correspondence between our nanosecond measurements and the published picosecond data suggests strongly that the intrinsic photochemical yield of all ferrous, hexacoordinate heme complexes approaches one. Third, the major differences between the isolated alpha and beta chains involve the rate of ligand migration to the solvent, kC----X and the extent of recombination from the second geminate state, C, as measured by the ratio kC----B/kC----X. Fourth, for both isolated chains and intact hemoglobin, the rate and equilibrium constants for the formation of the initial O2 geminate state starting from ligand in the solvent (i.e. kX----B and KX----B) are 5-10 times greater than the corresponding parameters for the formation of the first CO geminate state. Fifth, the rate-limiting step for NO, O2, and isonitrile binding to hemoglobin and its isolated subunits is ligand migration up to the initial geminate state (i.e. kX----B). In the case of CO binding, both migration to state B and iron-ligand bond formation (kB----A) affect the overall, bimolecular association rate constant.     

1H NMR study of labile proton exchange in the heme cavity as a probe for the potential ligand entry channel in myoglobin

The exchange rates of heme cavity histidine nitrogen-bound protons in horse and dog metcyanomyoglobins have been determined at 40 degrees C as a function of pH by 1H NMR spectroscopy. They were compared to the results reported for the sperm whale homologue [Cutnell, J. D., La Mar, G. N., & Kong, S. B. (1981) J. Am. Chem. Soc. 103, 3567-3572]. The rate profiles suggest that the exchange follows EX2-type kinetics, and the relative rate values favor a penetration model over a local unfolding model. It was found that the behavior of protons located on the proximal side of the heme is similar in the three proteins. The distal histidyl imidazole NH, however, shows a highly accelerated hydroxyl ion catalyzed rate in horse and dog myoglobins relative to that in sperm whale myoglobin. NMR spectral and relaxational characteristics of the assigned heme cavity protons indicate that the global geometry of the heme pocket is highly conserved in the ground-state structure of the three proteins. We propose a model that attributes the different distal histidine exchange behavior to the relative dynamic stability of the distal heme pocket in dog or horse myoglobin vs. sperm whale myoglobin. This model involves a dynamic equilibrium between a closed heme pocket as found in metaquomyoglobin [Takano, T. (1977) J. Mol. Biol. 110, 537-568] and an open pocket as found in phenylmetmyoglobin [Ringe, D., Petsko, G. A., Kerr, D. E., & Ortiz de Montellano, P. R. (1984) Biochemistry 23, 2-4].(ABSTRACT TRUNCATED AT 250 WORDS)     

Compressibility and uncoupling of cytochrome P450cam: high pressure FTIR and activity studies

The effect of the hydrostatic pressure on the CO ligand stretch vibration in cytochrome P450cam-CO bound with various substrates is studied by FTIR. The vibration frequency is linearily shifted to lower values with increasing pressure. The slope of the shift gives the isothermal compressibility of the heme pocket and is found to be related to the high-spin state content in an opposite direction to that previously observed from the pressure-induced shift of the Soret band. This opposite behaviour is explained by the dual effect of heme pocket water molecules both on the CO ligand and on electrostatic potentials produced by the protein at the distal side. The latter effect disturbs ligand-distal side contacts which are needed for a specific proton transfer in oxygen activation when dioxygen is the ligand. Their loss results in uncoupled H(2)O(2) formation.     

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)     

Crystal structures of ferrous and CO-, CN(-)-, and NO-bound forms of rat heme oxygenase-1 (HO-1) in complex with heme: structural implications for discrimination between CO and O2 in HO-1

Heme oxygenase (HO) catalyzes heme degradation by utilizing O(2) and reducing equivalents to produce biliverdin IX alpha, iron, and CO. To avoid product inhibition, the heme[bond]HO complex (heme[bond]HO) is structured to markedly increase its affinity for O(2) while suppressing its affinity for CO. We determined the crystal structures of rat ferrous heme[bond]HO and heme[bond]HO bound to CO, CN(-), and NO at 2.3, 1.8, 2.0, and 1.7 A resolution, respectively. The heme pocket of ferrous heme-HO has the same conformation as that of the previously determined ferric form, but no ligand is visible on the distal side of the ferrous heme. Fe[bond]CO and Fe[bond]CN(-) are tilted, whereas the Fe[bond]NO is bent. The structure of heme[bond]HO bound to NO is identical to that bound to N(3)(-), which is also bent as in the case of O(2). Notably, in the CO- and CN(-)-bound forms, the heme and its ligands shift toward the alpha-meso carbon, and the distal F-helix shifts in the opposite direction. These shifts allow CO or CN(-) to bind in a tilted fashion without a collision between the distal ligand and Gly139 O and cause disruption of one salt bridge between the heme and basic residue. The structural identity of the ferrous and ferric states of heme[bond]HO indicates that these shifts are not produced on reduction of heme iron. Neither such conformational changes nor a heme shift occurs on NO or N(3)(-) binding. Heme[bond]HO therefore recognizes CO and O(2) by their binding geometries. The marked reduction in the ratio of affinities of CO to O(2) for heme[bond]HO achieved by an increase in O(2) affinity [Migita, C. T., Matera, K. M., Ikeda-Saito, M., Olson, J. S., Fujii, H., Yoshimura, T., Zhou, H., and Yoshida, T. (1998) J. Biol. Chem. 273, 945-949] is explained by hydrogen bonding and polar interactions that are favorable for O(2) binding, as well as by characteristic structural changes in the CO-bound form.     

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.     

The X-ray structure determination of bovine carbonmonoxy hemoglobin at 2.1 A resoultion and its relationship to the quaternary structures of other hemoglobin crystal froms

Crystallographic studies of the intermediate states between unliganded and fully liganded hemoglobin (Hb) have revealed a large range of subtle but functionally important structural differences. Only one T state has been reported, whereas three other quaternary states (the R state, B state, and R2 or Y state) for liganded Hb have been characterized; other studies have defined liganded Hbs that are intermediate between the T and R states. The high-salt crystal structure of bovine carbonmonoxy (CO bovine) Hb has been determined at a resolution of 2.1 A and is described here. A detailed comparison with other crystallographically solved Hb forms (T, R, R2 or Y) shows that the quaternary structure of CO bovine Hb closely resembles R state Hb. However, our analysis of these structures has identified several important differences between CO bovine Hb and R state Hb. Compared with the R state structures, the beta-subunit N-terminal region has shifted closer to the central water cavity in CO bovine Hb. In addition, both the alpha- and beta-subunits in CO bovine Hb have more constrained heme environments that appear to be intermediate between the T and R states. Moreover, the distal pocket of the beta-subunit heme in CO bovine Hb shows significantly closer interaction between the bound CO ligand and the Hb distal residues Val 63(E11) and His 63(E7). The constrained heme groups and the increased steric contact involving the CO ligand and the distal heme residues relative to human Hb may explain in part the low intrinsic oxygen affinity of bovine Hb.