Combining transcriptomics and PBPK modeling indicates a primary role of hypoxia and altered circadian signaling in dichloromethane carcinogenicity in mouse lung and liver.

Dichloromethane (DCM) is a lung and liver carcinogen in mice at inhalation exposures≥2000ppm. The modes of action (MOA) of these responses have been attributed to formation of genotoxic, reactive metabolite(s). Here, we examined gene expression in lung and liver from female B6C3F1 mice exposed to 0, 100, 500, 2000, 3000 and 4000ppm DCM for 90days. We also simulated dose measures - rates of DCM oxidation to carbon monoxide (CO) in lung and liver and expected blood carboxyhemoglobin (HbCO) time courses with a PBPK model inclusive of both conjugation and oxidation pathways. Expression of large numbers of genes was altered at 100ppm with maximal changes in the numbers occurring by 500 or 2000ppm. Most changes in genes common to the two tissues were related to cellular metabolism and circadian clock. At the lower concentrations, the changes in metabolism-related genes were discordant - up in liver and down in lung. These processes included organelle biogenesis, TCA cycle, and respiratory electron transport. Changes in circadian cycle genes - primarily transcription factors - showed strong concentration-related response at higher concentrations (Arntl, Npas2, and Clock were down-regulated; Cry2, Wee1, Bhlhe40, Per3, Nr1d1, Nr1d2 and Dbp) were up-regulated with similar directionality in both tissues. Overall, persistently elevated HbCO from DCM oxidation appears to cause extended periods of hypoxia, leading to altered circadian coupling to cellular metabolism. The dose response for altered circadian processes correlates with the cancer outcome. We found no evidence of changes in genes indicative of responses to cytotoxic, DNA-reactive metabolites.

Blocking the gate to ligand entry in human hemoglobin.

His(E7) to Trp replacements in HbA lead to markedly biphasic bimolecular CO rebinding after laser photolysis. For isolated mutant subunits, the fraction of fast phase increases with increasing [CO], suggesting a competition between binding to an open conformation with an empty E7 channel and relaxation to blocked or closed, slowly reacting states. The rate of conformational relaxation of the open state is ∼18,000 s(-1) in α subunits and ∼10-fold faster in ß subunits, ∼175,000 s(-1). Crystal structures were determined for tetrameric α(WT)ß(Trp-63) HbCO, α(Trp-58)ß(WT) deoxyHb, and Trp-64 deoxy- and CO-Mb as controls. In Trp-63(E7) ßCO, the indole side chain is located in the solvent interface, blocking entry into the E7 channel. Similar blocked Trp-64(E7) conformations are observed in the mutant Mb crystal structures. In Trp-58(E7) deoxy-α subunits, the indole side chain fills both the channel and the distal pocket, forming a completely closed state. The bimolecular rate constant for CO binding, k'(CO), to the open conformations of both mutant Hb subunits is ∼80-90 µm(-1) s(-1), whereas k'(CO) for the completely closed states is 1000-fold slower, ∼0.08 µm(-1) s(-1). A transient intermediate with k'(CO) ≈ 0.7 µm(-1) s(-1) is observed after photolysis of Trp-63(E7) ßCO subunits and indicates that the indole ring blocks the entrance to the E7 channel, as observed in the crystal structures of Trp(E7) deoxyMb and ßCO subunits. Thus, either blocking or completely filling the E7 channel dramatically slows bimolecular binding, providing strong evidence that the E7 channel is the major pathway (≥90%) for ligand entry in human hemoglobin.

Carboxyhemoglobin levels in an unstable hemoglobin disorder (Hb Zürich): effect on phenotypic expression.

The affinity of Hb Zurich for carbon monoxide is approximately 65 times that of normal hemoglobin. The carboxyhemoglobin content in serum from individuals with Hb Zurich ranged from 3.9 to 6.7 percent in nine nonsmokers and from 9.8 to 19.7 percent in six smokers. Rates of hemolysis and hemoglobin denaturation were less in smokers than in nonsmokers, effects that may be secondary to the stabilization of Hb Zurich by carbon monoxide.

Five-coordinate H64Q neuroglobin as a ligand-trap antidote for carbon monoxide poisoning.

Carbon monoxide (CO) is a leading cause of poisoning deaths worldwide, with no available antidotal therapy. We introduce a potential treatment paradigm for CO poisoning, based on near-irreversible binding of CO by an engineered human neuroglobin (Ngb). Ngb is a six-coordinate hemoprotein, with the heme iron coordinated by two histidine residues. We mutated the distal histidine to glutamine (H64Q) and substituted three surface cysteines with less reactive amino acids to form a five-coordinate heme protein (Ngb-H64Q-CCC). This molecule exhibited an unusually high affinity for gaseous ligands, with a P50 (partial pressure of O2 at which hemoglobin is half-saturated) value for oxygen of 0.015 mmHg. Ngb-H64Q-CCC bound CO about 500 times more strongly than did hemoglobin. Incubation of Ngb-H64Q-CCC with 100% CO-saturated hemoglobin, either cell-free or encapsulated in human red blood cells, reduced the half-life of carboxyhemoglobin to 0.11 and 0.41 min, respectively, from ≥200 min when the hemoglobin or red blood cells were exposed only to air. Infusion of Ngb-H64Q-CCC to CO-poisoned mice enhanced CO removal from red blood cells, restored heart rate and blood pressure, increased survival, and was followed by rapid renal elimination of CO-bound Ngb-H64Q-CCC. Heme-based scavenger molecules with very high CO binding affinity, such as our mutant five-coordinate Ngb, are potential antidotes for CO poisoning by virtue of their ability to bind and eliminate CO.

Heme oxygenase-1 and carbon monoxide promote neovascularization after myocardial infarction by modulating the expression of HIF-1alpha, SDF-1alpha and VEGF-B.

Heme oxygenase-1 (HO-1), a known cytoprotective enzyme implicated also in the cell cycle regulation and angiogenesis, exerts many of its beneficial effects through carbon monoxide (CO). We studied the roles of HO-1 and CO in cardiac regeneration after myocardial infarction. Prior to coronary artery ligation, male Wistar rats were given either cobolt protoporphyrin IX to induce HO-1 or CO-donor methylene chloride. Cardiac regeneration was assessed by immunohistochemistry and confocal microscopy. CO significantly increased the accumulation of c-kit+ stem/progenitor cells into the infarct area and induced formation of new coronary arteries by promoting a substantial differentiation of c-kit+ cells into vascular smooth muscle cells (c-kit+/GATA6+ cells). Furthermore, CO increased proliferation of cardiomyocytes in the infarct border area at 4weeks post-infarction. This suggests proliferation of newly formed cardiomyocytes derived from c-kit+ cells as 10% of c-kit+ cells expressed early cardiac marker Nkx2.5. Increased expression of hypoxia-inducible factor-1alpha (HIF-1alpha), stromal cell derived factor-1alpha (SDF-1alpha) and vascular endothelial growth factor-B (VEGF-B) were found in the infarct areas of CO-donor pretreated hearts suggesting that these factors potentially promoted the migration of c-kit+ cells into the infarct area and subsequent vasculogenesis and myocardial regeneration by CO. HO-1 increased both capillary and vascular densities, while only a small increase of c-kit+ cells was found. HO-1 upregulated SDF-1alpha, but did not have effect on HIF-1alpha and VEGF-B. In conclusion, HO-1 and CO have differential roles and mechanisms of action in cardiac regeneration. Modulation of the HO-1/CO axis may provide a novel tool for the repair of cardiac injury.

Quaternary structure of carbonmonoxyhemoglobins in solution: structural changes induced by the allosteric effector inositol hexaphosphate.

We have applied the residual dipolar coupling (RDC) method to investigate the solution quaternary structures of (2)H- and (15)N-labeled human normal adult recombinant hemoglobin (rHb A) and a low-oxygen-affinity mutant recombinant hemoglobin, rHb(alpha96Val-->Trp), both in the carbonmonoxy form, in the absence and presence of an allosteric effector, inositol hexaphosphate (IHP), using a stretched polyacrylamide gel as the alignment medium. Our recent RDC results [Lukin, J. A., Kontaxis, G., Simplaceanu, V., Yuan, Y., Bax, A., and Ho, C. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 517-520] indicate that the quaternary structure of HbCO A in solution is a dynamic ensemble between two previously determined crystal structures, R (crystals grown under high-salt conditions) and R2 (crystals grown under low-salt conditions). On the basis of a comparison of the geometric coordinates of the T, R, and R2 structures, it has been suggested that the oxygenation of Hb A follows the transition pathway from T to R and then to R2, with R being the intermediate structure [Srinivasan, R., and Rose, G. D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11113-11117]. The results presented here suggest that IHP can shift the solution quaternary structure of HbCO A slightly toward the R structure. The solution quaternary structure of rHbCO(alpha96Val-->Trp) in the absence of IHP is similar to that of HbCO A in the presence of IHP, consistent with rHbCO(alpha96Val-->Trp) having an affinity for oxygen lower than that of Hb A. Moreover, IHP has a much stronger effect in shifting the solution quaternary structure of rHbCO(alpha96Val-->Trp) toward the R structure and toward the T structure, consistent with IHP causing a more pronounced decrease in its oxygen affinity. The results presented in this work, as well as other results recently reported in the literature, clearly indicate that there are multiple quaternary structures for the ligated form of hemoglobin. These results also provide new insights regarding the roles of allosteric effectors in regulating the structure and function of hemoglobin. The classical two-state/two-structure allosteric mechanism for the cooperative oxygenation of hemoglobin cannot account for the structural and functional properties of this protein and needs to be revised.

COHbC and COHbS crystallize in the R2 quaternary state at neutral pH in the presence of PEG 4000.

Human hemoglobin binds oxygen cooperatively and functions as a tetramer composed of two identical alphabeta heterodimers. While human hemoglobin is the best characterized allosteric protein, the quaternary R (oxygenated or liganded) to T (deoxygenated) structural transition remains controversial. The R2 state has been postulated to represent either an intermediate or final quaternary state elicited by ligand binding. However, the biological relevance of the R2 state has been questioned as it has not been observed crystallographically under physiological conditions. The high-resolution R2 quaternary structures of human COHbC (betaE6K) and COHbS (betaE6V) are reported at neutral pH and low ionic strength using PEG 4000 as a precipitant. Crystals of COHbC, COHbS and their mixtures are isomorphous, indicating that they share the same tertiary and quaternary structures. In contrast, oxyHbA or COHbA did not yield crystals at neutral pH under similar conditions. Solubility studies and modeling suggest that at neutral pH and low ionic strength the beta6 mutant hemoglobins crystallize (betaK6 > betaV6) as a result of more favorable lattice contacts.

Structure of mutant human carbonmonoxyhemoglobin C (betaE6K) at 2.0 A resolution.

Previous studies have demonstrated that in vitro crystallization of R-state liganded hemoglobin C (HbC), a naturally occurring mutant human hemoglobin (betaE6K), in high-phosphate buffer solutions provides a potential model system for the intracellular crystallization of HbC associated with chronic hemolytic anemia in CC disease. The first high-resolution crystal structure of liganded HbC is reported here. HbC was crystallized from high phosphate and the structure of the carbonmonoxy-liganded R-state form was refined at 2.0 A resolution. Crystals exhibit diffraction consistent with the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = 54.16, c = 195.30 A. The structure was solved by difference Fourier techniques and refinement by simulated annealing and restrained least-squares yielded a final R of 0.183 and an R(free) of 0.238 for all 19,382 unique reflections. The side chain of betaK6 exhibits very weak electron density consistent with significant mobility within the crystalline lattice. The highly dynamic nature of the side chain could potentially support a number of specific polar interactions that might reduce the barrier to crystallization and thus result in enhanced crystallization kinetics for HbC relative to HbA. Specifically, the NZ atom of the BK6 side chain could participate in an amino-aromatic hydrogen bond with the pi-electron cloud of betaH116 in a symmetry-related tetramer. BetaK6 NZ might also interact with the main-chain carbonyl O atom of betaH117 and the carboxylate group of betaE22 from a symmetry-related tetramer.

The mutation beta 99 Asp-Tyr stabilizes Y--a new, composite quaternary state of human hemoglobin.

Carbonmonoxy hemoglobin Ypsilanti (beta 99 Asp-Tyr) exhibits a quaternary form distinctly different from any structures previously observed for human hemoglobins. The relative orientation of alpha beta dimers in the new quaternary form lies well outside the range of values observed for normal unliganded and liganded tetramers (Baldwin, J., Chothia, C., J. Mol. Biol. 129:175-220, 1979). Despite this large quaternary structural difference between carbonmonoxy hemoglobin Ypsilanti and the two canonical structures, the new quaternary structure's hydrogen bonding interactions in the "switch" region, and packing interactions in the "flexible joint" region, show noncovalent interactions characteristic of the alpha 1 beta 2 contacts of both unliganded and liganded normal hemoglobins. In contrast to both canonical structures, the beta 97 histidine residue in carbonmonoxy hemoglobin Ypsilanti is disengaged from quaternary packing interactions that are generally believed to enforce two-state behavior in ligand binding. These features of the new quaternary structure, denoted Y, may therefore be representative of quaternary states that occur transiently along pathways between the normal unliganded, T, and liganded, R, hemoglobin structures.

Dynamic properties of some beta-chain mutant hemoglobins.

The thermal behavior of the Soret band relative to the carbonmonoxy derivatives of some beta-chain mutant hemoglobins is studied in the temperature range 300-10 K and compared to that of wild-type carbonmonoxy hemoglobin. The band profile at various temperatures is modeled as a Voigt function that accounts for homogeneous broadening and for the coupling with high- and low-frequency vibrational modes, while inhomogeneous broadening is taken into account with a gaussian distribution of purely electronic transition frequencies. The various contributions to the over-all bandwidth are singled out with this analysis and their temperature dependence, in turn, gives information on structural and dynamic properties of the system studied. In the wild-type and mutant hemoglobins, the values of homogeneous bandwidth and of the coupling constants to high-frequency vibrational modes are not modified with respect to natural human hemoglobin, thus indicating that the local electronic and vibrational properties of the heme-CO complex are not altered by the recombinant procedures. On the contrary, differences in the protein dynamic behavior are observed. The most relevant are those relative to the "polar isosteric" beta Val-67(E11)-->Thr substitution, localized in the heme pocket, which results in decreased coupling with low-frequency modes and increased anharmonic motions. Mutations involving residue beta Lys-144(Hc1) at the C-terminal and residue beta Cys-112(G14) at the alpha 1 beta 1 interface have a smaller effect consisting in an increased coupling with low-frequency modes. Mutations at the beta-N-terminal and at the alpha 1 beta 2 interface have no effect on the dynamic properties of the same heme pocket.

Human carboxyhemoglobin at 2.2 A resolution: structure and solvent comparisons of R-state, R2-state and T-state hemoglobins.

The three-dimensional structure and associated solvent of human carboxyhemoglobin at 2.2 A resolution are compared with other R-state and T-state human hemoglobin structures. The crystal form is isomorphous with that of the 2.7 A structure of carboxyhemoglobin reported earlier [Baldwin (1980). J. Mol. Biol. 136, 103-128], whose coordinates were used as a starting model, and with the 2.2 A structure described in an earlier report [Derewenda et al. (1990). J. Mol. Biol. 211, 515-519]. During the course of the refinement, a natural mutation of the alpha-subunit, A53S, was discovered that forms a new crystal contact through a bridging water molecule. The protein structure shows a significant difference between the alpha and beta heme geometries, with Fe-C-O angles of 125 and 162 degrees, respectively. The carboxyhemoglobin is compared with other fully ligated R-state human hemoglobins [Baldwin (1980). J. Mol. Biol. 136, 103-128; Shaanan (1983). J. Mol. Biol. 195, 419-422] with the R2-state hemoglobin [Silva et al. (1992). J. Biol. Chem. 267, 17248-17256] and with T-state deoxyhemoglobin [Fronticelli et al. (1994). J. Biol. Chem. 269, 23965-23969]. The structure is similar to the earlier reported R-state structures, but there are differences in many side-chain conformations, the associated water structure and the presence and the position of a phosphate ion. The quaternary changes between the R-state carboxyhemoglobin and the R2-state and T-state structures are in general consistent with those reported in the earlier structures. The location of 238 water molecules and a phosphate ion in the carboxyhemoglobin structure allows the first comparison of the solvent structures of the R-state and T-state structures. Distinctive hydration patterns for each of the quaternary structures are observed, but a number of conserved water molecule binding sites are found that are independent of the conformational state of the protein.

The carbon monoxide derivative of human hemoglobin carrying the double mutation LeuB10-->Tyr and HisE7-->Gln on alpha and beta chains probed by infrared spectroscopy.

The fine structural properties of the distal heme pocket have been probed by infrared spectroscopy of ferrous carbon monoxy human hemoglobin mutants carrying the mutations LeuB10-->Tyr and HisE7-->Gln on the alpha, beta, and both chains, respectively. The stretching frequency of iron-bound carbon monoxide occurs as a single broad band around 1943 cm(-1) in both the alpha and the beta mutated chains. Such a frequency value indicates that no direct hydrogen bonding exists between the bound CO molecule and the TyrB10 phenolic oxygen, at variance with other naturally occurring TyrB10, GlnE7 nonvertebrate hemoglobins. The rates of carbon monoxide release have been determined for the first time by a Fourier transform infrared spectroscopy stopped-flow technique that allowed us to single out the heterogeneity in the kinetics of CO release in the alpha and beta chains for the mutated proteins and for native HbA. The rates of CO release are 15- to 20-fold faster for the mutated alpha or beta chains with respect to the native ones consistent with the lack of distal stabilization on the iron-bound CO molecule. The present results demonstrate that residues in key topological positions (namely E7 and B10) for the distal steric control of the iron-bound ligand are not interchangeable among hemoglobins from different species.

Polymerization of recombinant Hb S-Kempsey (deoxy-R state) and Hb S-Kansas (oxy-T state).

In order to investigate the role of the R (relaxed) to T (tense) structural transition in facilitating polymerization of deoxy-Hb S, we have engineered and expressed two Hb S variants which destabilize either T state (Hb S-Kempsey, alpha 2 beta 2 Val-6,Asn-99) or R state structures (Hb S-Kansas, alpha 2 beta 2 Val-6, Thr-102). Polymerization of deoxy-Hb S-Kempsey, which shows high oxygen affinity and increased dimer dissociation, required about 2- and 6-fold higher hemoglobin concentrations than deoxy-Hb S for polymerization in low and high phosphate concentrations, and its kinetic pattern of polymerization was biphasic. In contrast, oxy- or CO Hb S-Kansas, which shows low oxygen affinity and increased dimer dissociation, polymerized at a slightly higher critical concentration than that required for polymerization of deoxy-Hb S in both low and high phosphate buffers. Polymerization of oxy- and CO Hb S-Kansas was linear and showed no delay time, which is similar to oversaturated oxy- or CO Hb S. These results suggest that nuclei formation, which occurs during the delay time prior to deoxy-Hb S polymerization, does not occur in T state oxy-Hb S-Kansas, even though the critical concentration for polymerization of T state oxy-Hb S-Kansas is similar to that of T state deoxy-Hb S.

Structures of the deoxy and CO forms of haemoglobin from Dasyatis akajei, a cartilaginous fish.

The three-dimensional structures of the deoxy- and carbonmonoxyhaemoglobin (Hb) from Dasyatis akajei, a stingray, have been determined at 1.6 and 1.9 A resolution, respectively. This is one of the most distantly related vertebrate Hbs to human HbA. Both structures resemble the respective forms of HbA, indicating that the alpha2beta2-type tetramer and the mode of the quaternary structure change are common to Hbs of jawed vertebrates. Larger deviations between D. akajei Hb and human HbA are observed in various parts of the molecule, even in the E and F helices. Significant mutations and/or conformational changes are also observed around the haems, in the C-terminal region of the beta subunit, in the alpha1beta2 interface and in the organic phosphate-binding site of HbA. Despite these structural differences, the oxygen affinity, haem-haem interaction, Bohr effect and organic phosphate effect of D. akajei Hb are all only moderately reduced. Compared with human HbA, the overall r.m.s. deviation of main-chain atoms in the helical regions of bony fish Hbs is smaller than that of D. akajei Hb.

Crystallographic, molecular modeling, and biophysical characterization of the valine beta 67 (E11)-->threonine variant of hemoglobin.

The crystal structure of the mutant deoxyhemoglobin in which the beta-globin Val67(E11) has been replaced with threonine [Fronticelli et al. (1993) Biochemistry 32, 1235-1242] has been determined at 2.2 A resolution. Prior to the crystal structure determination, molecular modeling indicated that the Thr67(E11) side chain hydroxyl group in the distal beta-heme pocket forms a hydrogen bond with the backbone carbonyl of His63(E7) and is within hydrogen-bonding distance of the N delta of His63(E7). The mutant crystal structure indicates only small changes in conformation in the vicinity of the E11 mutation confirming the molecular modeling predictions. Comparison of the structures of the mutant beta-subunits and recombinant porcine myoglobin with the identical mutation [Cameron et al. (1993) Biochemistry 32, 13061-13070] indicates similar conformations of residues in the distal heme pocket, but there is no water molecule associated with either of the threonines of the beta-subunits. The introduction of threonine into the distal heme pocket, despite having only small perturbations in the local structure, has a marked affect on the interaction with ligands. In the oxy derivative there is a 2-fold decrease in O2 affinity [Fronticelli et al. (1993) Biochemistry 32, 1235-1242], and the rate of autoxidation is increased by 2 orders of magnitude. In the CO derivative the IR spectrum shows modifications with respect to that of normal human hemoglobin, suggesting the presence of multiple CO conformers. In the nitrosyl derivative an interaction with the O gamma atom of Thr67(E11) is probably responsible for the 10-fold increase in the rate of NO release from the beta-subunits. In the aquomet derivative there is a 6-fold decrease in the rate of hemin dissociation suggesting an interaction of the Fe-coordinated water with the O gamma of Thr67(E11).