Topology and thermodynamics of gaseous ligands diffusion paths in human neuroglobin

The physiological role of recently discovered human neuroglobin (Ngb) is still unknown. Sound hypothesis says that it protects brain during hypoxia. In this paper the advanced potential of mean force by implicit ligand sampling (PMF/ILS) method is used to study the free energy landscape of Ngb for O₂, NO and CO ligands. The multiple diffusion paths are discovered and four ligand binding cavities are determined. The data show that certain regions are easily accessible by O₂ and NO but are protected from CO. Free energy landscapes provide realistic data for stochastic models of ligand diffusion in proteins.     

Reversible hexa- to penta-coordination of the heme Fe atom modulates ligand binding properties of neuroglobin and cytoglobin

Neuroglobin (Ngb) and cytoglobin (Cygb) are two recently discovered intracellular members of the vertebrate hemoglobin (Hb) family. Ngb, predominantly expressed in nerve cells, is of ancient evolutionary origin and is homologous to nerve-globins of invertebrates. Cygb, present in many different tissues, shares common ancestry with myoglobin (Mb) and can be traced to early vertebrate evolution. Ngb is held to facilitate O2 diffusion to the mitochondria and to protect neuronal cells from hypoxic-ischemic insults, may be an oxidative stress-responsive sensor protein for signal transduction, and may carry out enzymatic activities, such as NO/O2 scavenging. Cygb is linked to collagen synthesis, may provide O2 for enzymatic reactions, and may be involved in a ROS(NO)-signaling pathway(s). Ngb and Cgb display the classical three-over-three alpha-helical fold of Hb and Mb, and are endowed with a hexa-coordinate heme-Fe atom, in their ferrous and ferric forms, having the heme distal HisE7 residue as the endogenous ligand. Reversible hexa- to penta-coordination of the heme Fe atom modulates ligand binding properties of Ngb and Cygb. Moreover, Ngb and Cygb display a tunnel/cavity system within the protein matrix held to facilitate ligand channeling to/from the heme, multiple ligand copies storage, multi-ligand reactions, and conformational transitions supporting ligand binding.     

Structural characterization of the proximal and distal histidine environment of cytoglobin and neuroglobin

Cytoglobin (Cgb) and neuroglobin (Ngb) are the first examples of hexacoordinated globins from humans and other vertebrates in which a histidine (His) residue at the sixth position of the heme iron is an endogenous ligand in both the ferric and ferrous forms. Static and time-resolved resonance Raman and FT-IR spectroscopic techniques were applied in examining the structures in the heme environment of these globins. Picosecond time-resolved resonance Raman (ps-TR3) spectroscopy of transient five-coordinate heme species produced by the photolysis of carbon monoxide (CO) adducts of Cgb and Ngb showed Fe-His stretching (nu(Fe-His)) bands at 229 and 221 cm(-1), respectively. No time-dependent shift in the nu(Fe-His) band of Cgb and Ngb was detected in the 20-1000 ps time domain, in contrast to the case of myoglobin (Mb). These spectroscopic data, combined with previously reported crystallographic data, suggest that the structure of the heme pocket in Cgb and Ngb is altered upon CO binding in a manner different from that of Mb and that the scales of the structural alteration are different for Cgb and Ngb. The structural property of the heme distal side of the ligand-bound forms was investigated by observing the sets of (nu(Fe-CO), nu(C-O), delta(Fe-C-O)) and (nu(Fe-NO), nu(N-O), delta(Fe-N-O)) for the CO and nitric oxide (NO) complexes of Cgb and Ngb. A comparison of the spectra of some distal mutants of Cgb (H81A, H81V, R84A, R84K, and R84T) and Ngb (H64A, H64V, K67A, K67R, and K67T) showed that the CO adducts of Cgb and Ngb contained three conformers and that the distal His (His81 in Cgb and His64 in Ngb) mainly contributes to the interconversion of the conformers. These structural characteristics of Cgb and Ngb are discussed in relation to their ligand binding and physiological properties.     

Electron transfer function versus oxygen delivery: a comparative study for several hexacoordinated globins across the animal kingdom

Caenorhabditis elegans globin GLB-26 (expressed from gene T22C1.2) has been studied in comparison with human neuroglobin (Ngb) and cytoglobin (Cygb) for its electron transfer properties. GLB-26 exhibits no reversible binding for O(2) and a relatively low CO affinity compared to myoglobin-like globins. These differences arise from its mechanism of gaseous ligand binding since the heme iron of GLB-26 is strongly hexacoordinated in the absence of external ligands; the replacement of this internal ligand, probably the E7 distal histidine, is required before binding of CO or O(2) as for Ngb and Cygb. Interestingly the ferrous bis-histidyl GLB-26 and Ngb, another strongly hexacoordinated globin, can transfer an electron to cytochrome c (Cyt-c) at a high bimolecular rate, comparable to those of inter-protein electron transfer in mitochondria. In addition, GLB-26 displays an unexpectedly rapid oxidation of the ferrous His-Fe-His complex without O(2) actually binding to the iron atom, since the heme is oxidized by O(2) faster than the time for distal histidine dissociation. These efficient mechanisms for electron transfer could indicate a family of hexacoordinated globin which are functionally different from that of pentacoordinated globins.     

The heme environment of mouse neuroglobin. Evidence for the presence of two conformations of the heme pocket

Neuroglobin (Ngb) is a newly discovered oxygen-binding heme protein that is primarily expressed in the brain of humans and other vertebrates. To characterize the structure/function relationships of this new heme protein, we have used resonance Raman spectroscopy to determine the structure of the heme environment in Ngb from mice. In the Fe(2+)CO complex, two conformations of the Fe-CO unit are present, one of which arises from an open conformation of the heme pocket in which the CO is not interacting with any nearby residue, and the other arises from a closed conformation where a positively charged residue near the CO group stabilizes the complex. For the Fe(2+)O(2) complex, we detect a single nu(Fe-OO) stretching mode at a frequency similar to that of oxymyoglobins and oxyhemoglobins of vertebrates (571 cm(-1)). Based on the Fe-C-O frequencies of the closed conformation of Ngb, a highly polar distal environment is indicated from which the O(2) off-rate is predicted to be lower than that of Mb. In the absence of exogenous ligands, a heme pocket residue coordinates to the heme iron, forming a six-coordinate complex, thereby predicting a low on-rate for exogenous ligands. These structural properties of the heme pocket of Ngb are discussed with respect to its proposed in vivo oxygen delivery function.     

Molecular dynamics simulation of deoxy and carboxy murine neuroglobin in water

Globins are respiratory proteins that reversibly bind dioxygen and other small ligands at the iron of a heme prosthetic group. Hemoglobin and myoglobin are the most prominent members of this protein family. Unexpectedly a few years ago a new member was discovered and called neuroglobin (Ngb), being predominantly expressed in the brain. Ngb is a single polypeptide of 151 amino acids and despite the small sequence similarity with other globins, it displays the typical globin fold. Oxygen, nitric oxide, or carbon monoxide can displace the distal histidine which, in ferrous Ngb as well as in ferric Ngb, is bound to the iron, yielding a reversible adduct. Recent crystallographic data on carboxy Ngb show that binding of an exogenous ligand is associated to structural changes involving heme sliding and a topological reorganization of the internal cavities; in particular, the huge internal tunnel that connects the bulk with the active site, peculiar to Ngb, is heavily reorganized. We report the results of extended (90 ns) molecular dynamics simulations in water of ferrous deoxy and carboxy murine neuroglobin, which are both coordinated on the distal site, in the latter case by CO and in the former one by the distal His(64)(E7). The long timescale of the simulations allowed us to characterize the equilibrated protein dynamics and to compare protein structure and dynamical behavior coupled to the binding of an exogenous ligand. We have characterized the heme sliding motion, the topological reorganization of the internal cavities, the dynamics of the distal histidine, and particularly the conformational change of the CD loop, whose flexibility depends ligand binding.     

Recognition and discrimination of gases by the oxygen-sensing signal transducer protein HemAT as revealed by FTIR spectroscopy

The determination of ligand binding properties is a key step in our understanding of gas sensing and discrimination by gas sensory proteins. HemAT is a newly discovered signal transducer heme protein that recognizes O(2) and discriminates against other gases such as CO and NO. We have used FTIR spectroscopy on CO- and NO-bound sensor domain HemAT and sensor domain distal mutants Y70F, T95A, R91A, and L92A to gain insight into the structure of the iron-bound ligand at ambient temperature. These mutations were designed to perturb the electrostatic field near the iron-bound gaseous ligand and also allow us to investigate the communication pathway between the distal residues of the protein and the heme. We show the formation of both H-bonded and non-H-bonded conformations in the CO-bound forms. In addition, we report the presence of multiple conformations in the NO-bound forms. Such distal H-bonding is crucial for ligand binding and activation by the heme. The comparison of the O(2), NO, and CO data demonstrates that Thr95 and Tyr70 are crucial for ligand recognition and discrimination and, thus, for specific sensing of gases, and L92 is crucial for controlling the conformational changes of the Thr95 and Tyr70 residues upon NO binding.     

Kinetic studies of the reactions of O(2) and NO with reduced Thermus thermophilus ba(3) and bovine aa(3) using photolabile carriers

The reactions of molecular oxygen (O(2)) and nitric oxide (NO) with reduced Thermus thermophilus (Tt) ba(3) and bovine heart aa(3) were investigated by time-resolved optical absorption spectroscopy to establish possible relationships between the structural diversity of these enzymes and their reaction dynamics. To determine whether the photodissociated carbon monoxide (CO) in the CO flow-flash experiment affects the ligand binding dynamics, we monitored the reactions in the absence and presence of CO using photolabile O(2) and NO complexes. The binding of O(2)/NO to reduced ba(3) in the absence of CO occurs with a second-order rate constant of 1×10(9)M(-1)s(-1). This rate is 10-times faster than for the mammalian enzyme, and which is attributed to structural differences in the ligand channels of the two enzymes. Moreover, the O(2)/NO binding in ba(3) is 10-times slower in the presence of the photodissociated CO while the rates are the same for the bovine enzyme. This indicates that the photodissociated CO directly or indirectly impedes O(2) and NO access to the active site in Tt ba(3), and that traditional CO flow-flash experiments do not accurately reflect the O(2) and NO binding kinetics in ba(3). We suggest that in ba(3) the binding of O(2) (NO) to heme a(3)(2+) causes rapid dissociation of CO from Cu(B)(+) through steric or electronic effects or, alternatively, that the photodissociated CO does not bind to Cu(B)(+). These findings indicate that structural differences between Tt ba(3) and the bovine aa(3) enzyme are tightly linked to mechanistic differences in the functions of these enzymes. This article is part of a Special Issue entitled: Respiratory Oxidases.     

The Structure of Neuroglobin at High Xe and Kr Pressure Reveals Partial Conservation of Globin Internal Cavities

Neuroglobin (Ngb) is a hexacoordinate globin expressed in the brain of vertebrates. Ferrous Ngb binds dioxygen with high affinity and the O₂ adduct is able to scavenge NO. Convincing in vitro and in vivo data indicate that Ngb is involved in neuroprotection during hypoxia and ischemia. The 3D structure of Ngb reveals the presence of a wide internal cavity connecting its heme active site with the bulk. To explore the role of this “tunnel” in the control of ligand binding, we determined the structure of metNgb and NgbCO equilibrated with Xe or Kr. We show four docking sites for Xe (only two for Kr); two of the four Xe sites are within the large cavity. They are only partially conserved in globins, since the two proximal Xe sites identified in myoglobin (Xe1 and Xe2) are absent in Ngb, as well as in cytoglobin. The Xe docking sites in Ngb map a pathway within the protein matrix, leading to the heme, which becomes more accessible in the ligand-bound species. This may be of significance in connection with the redox chemistry that may be the primary function of this hexacoordinate globin.     

Electron paramagnetic resonance- (EPR-) resolved kinetics of cryogenic nitric oxide recombination to cytochrome c oxidase and myoglobin.

By the electron paramagnetic resonance (EPR) technique, recovery kinetics for nitric oxide (NO) to heme following cryogenic photolysis were studied for the nitrosylferrocytochrome a3 center in cytochrome c oxidase and for myoglobin. The recovery was nonexponential, as has been observed in previous cryogenic CO and O2 rebinding to heme systems. NO rebinding to heme a3 started near a temperature of 50 K and was related to a distribution of thermal activation energies. At the peak of the distribution the activation energy was 3.1 kcal/mol, and the preexponential in the recovery rate was 10(9.9) s-1. For recovery of NO back to the a3 heme, the activation energy was threefold less than that for CO where CO binds to nearby Cua3 following photolysis from heme a3, but was larger than the activation energy for CO, O2, and probably NO rebinding to myoglobin. NO ligand rebinding to myoglobin occurred at a temperature as low as 15 K and in a temperature regime where tunneling could occur. However, the rate of NO rebinding to myoglobin did increase with temperature in the 15-25 K range.     

Structural basis of human cytoglobin for ligand binding

Cytoglobin (Cgb), a newly discovered member of the vertebrate globin family, binds O(2) reversibly via its heme, as is the case for other mammalian globins (hemoglobin (Hb), myoglobin (Mb) and neuroglobin (Ngb)). While Cgb is expressed in various tissues, its physiological role is not clearly understood. Here, the X-ray crystal structure of wild type human Cgb in the ferric state at 2.4A resolution is reported. In the crystal structure, ferric Cgb is dimerized through two intermolecular disulfide bonds between Cys38(B2) and Cys83(E9), and the dimerization interface is similar to that of lamprey Hb and Ngb. The overall backbone structure of the Cgb monomer exhibits a traditional globin fold with a three-over-three alpha-helical sandwich, in which the arrangement of helices is basically the same among all globins studied to date. A detailed comparison reveals that the backbone structure of the CD corner to D helix region, the N terminus of the E-helix and the F-helix of Cgb resembles more closely those of pentacoordinated globins (Mb, lamprey Hb), rather than hexacoordinated globins (Ngb, rice Hb). However, the His81(E7) imidazole group coordinates directly to the heme iron as a sixth axial ligand to form a hexcoordinated heme, like Ngb and rice Hb. The position and orientation of the highly conserved residues in the heme pocket (Phe(CD1), Val(E11), distal His(E7) and proximal His(F8)) are similar to those of other globin proteins. Two alternative conformations of the Arg84(E10) guanidium group were observed, suggesting that it participates in ligand binding to Cgb, as is the case for Arg(E10) of Aplysia Mb and Lys(E10) of Ngb. The structural diversities and similarities among globin proteins are discussed with relevance to molecular evolutionary relationships.     

Interdomain interactions within the two-component heme-based sensor DevS from Mycobacterium tuberculosis

DevS is the sensor of the DevS-DevR two-component regulatory system of Mycobacterium tuberculosis. This system is thought to be responsible for initiating entrance of this bacterium into the nonreplicating persistent state in response to NO and anaerobiosis. DevS is modular in nature and consists of two N-terminal GAF domains and C-terminal histidine kinase and ATPase domains. The first GAF domain (GAF A) binds heme, and this cofactor is thought to be responsible for sensing environmental stimuli, but the function of the second GAF domain (GAF B) is unknown. Here we report the RR characterization of full-length DevS (FL DevS) as well as truncated proteins consisting of the single GAF A domain (GAF A DevS) and both GAF domains (GAF A/B) in both oxidation states and bound to the exogenous ligands CO, NO, and O2. The results indicate that the GAF B domain increases the specificity with which the distal heme pocket of the GAF A domain interacts with CO and NO as opposed to O2. Specifically, while two comparable populations of CO and NO adducts are observed in GAF A DevS, only one of these two conformers is present in significant concentration in the GAF A/B and FL DevS proteins. In contrast, hydrogen bond interactions at the bound oxygen in the oxy complexes are conserved in all DevS constructs. The comparison of the data obtained with the O2 complexes with those of the CO and NO complexes suggests a model for ligand discrimination which relies on a specific hydrogen-bonding network with bound O2. It also suggests that interactions between the two GAF domains are responsible for transduction of structural changes at the heme domain that accompany ligand binding/dissociation to modulate activity at the kinase domain.     

A Spectroscopic Study of Structural Heterogeneity and Carbon Monoxide Binding in Neuroglobin

Neuroglobin (Ngb) is a small globular protein that binds diatomic ligands like oxygen, carbon monoxide (CO) and nitric oxide at a heme prosthetic group. We have performed FTIR spectroscopy in the infrared stretching bands of CO and flash photolysis with monitoring in the electronic heme absorption bands to investigate structural heterogeneity at the active site of Ngb and its effects on CO binding and migration at cryogenic temperatures. Four CO stretching bands were identified; they correspond to discrete conformations that differ in structural details and CO binding properties. Based on a comparison of bound-state and photoproduct IR spectra of the wild-type protein, Ngb distal pocket mutants and myoglobin, we have provided structural interpretations of the conformations associated with the different CO bands. We have also studied ligand migration to the primary docking site, B. Rebinding from this site is governed by very low enthalpy barriers (∼1 kJ/mol), indicating an extremely reactive heme iron. Moreover, we have observed ligand migration to a secondary docking site, C, from which CO rebinding involves higher enthalpy barriers.     

Identification of protein radicals formed in the human neuroglobin-H2O2 reaction using immuno-spin trapping and mass spectrometry

Neuroglobin (Ngb) is a recently discovered protein that shows only minor sequence similarity with myoglobin and hemoglobin but conforms to the typical 3-over-3 alpha-helical fold characteristic of vertebrate globins. An intriguing feature of Ngb is its heme hexacoordination in the absence of external ligands, observed both in the ferrous and in the ferric (met) forms. In Ngb, the imidazole of a histidine residue (His-64) in the distal position, above the heme plane, provides the sixth coordination bond. In this work, a valine residue was introduced at position 64 (H64V variant) to clarify the possible role(s) of the distal residue in protecting the heme iron of Ngb from attack by strong oxidants. SDS-PAGE analyses revealed that the oxidation of the H64V variant of metNgb by H 2O 2 resulted in the formation of dimeric and trimeric products in contrast to the native protein. Dityrosine cross-links were shown by their fluorescence to be present in the oligomeric products. When the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was included in the reaction mixture, nitrone adducts were detected by immuno-spin trapping. The specific location of the DMPO adducts on the H64V variant protein was determined by a mass spectrometry method that combines off-line immuno-spin trapping and chromatographic procedures. This method revealed Tyr-88 to be the site of modification by DMPO. The presence of His-64 in the wild-type protein results in the nearly complete loss of detectable radical adducts. Together, the data support the argument that wild-type Ngb is protected from attack by H 2O 2 by the coordinated distal His.     

Control of heme reactivity by diffusion: structural basis and functional characterization in hemoglobin mutants.

The effect of mutagenesis on O(2), CO, and NO binding to mutants of human hemoglobin, designed to modify some features of the reactivity that hinder use of hemoglobin solutions as blood substitute, has been extensively investigated. The kinetics may be interpreted in the framework of the Monod-Wyman-Changeux two-state allosteric model, based on the high-resolution crystallographic structures of the mutants and taking into account the control of heme reactivity by the distal side mutations. The mutations involve residues at topological position B10 and E7, i.e., Leu (B10) to Tyr and His (E7) to Gln, on either the alpha chains alone (yielding the hybrid tetramer Hbalpha(YQ)), the beta chains alone (hybrid tetramer Hbbeta(YQ)), or both types of chains (Hb(YQ)). Our data indicate that the two mutations affect ligand diffusion into the pocket, leading to proteins with low affinity for O(2) and CO, and especially with reduced reactivity toward NO, a difficult goal to achieve. The observed kinetic heterogeneity between the alpha(YQ) and beta(YQ) chains in Hb(YQ) has been rationalized on the basis of the three-dimensional structure of the active site. Furthermore, we report for the first time an experiment of partial CO binding, selective for the beta chains, to high salt crystals of the mutant Hb(YQ) in the T-state; these crystallographic data may be interpreted as "snapshots" of the initial events possibly occurring on ligand binding to the T-allosteric state of this peculiar mutant Hb.     

Distal mutation modulates the heme sliding in mouse neuroglobin investigated by molecular dynamics simulation

Neuroglobin (Ngb), a hexa‐coordinated hemoprotein primarily expressed in the brain and retina, is thought to be involved in neuroprotection and signal transduction. Ngb can reversibly bind small ligands such as O₂ and CO to the heme iron by replacing the distal histidine which is bound to the iron as the endogenous ligand. In this work, molecular dynamics (MD) simulations were performed to investigate the functionally related structural properties and dynamical characteristics in carboxy mouse neuroglobin and three distal mutants including single mutants H64V, K67T and double mutant H64V/K67T. MD simulations suggest that the heme sliding motion induced by the binding of exogenous ligand is affected by the distal mutation obviously. Accompanying changes in loop flexibility and internal cavities imply the structural rearrangement of Ngb. Moreover, the solvent accessibility of heme and some crucial residues are influenced revealing an interactive network on the distal side. The work elucidates that the key residues K67 at E10 and H64 at E7 are significant in modulating the heme sliding and hence the structural and physiological function of Ngb. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Pulmonary diffusing capacities for nitric oxide and carbon monoxide determined by rebreathing in dogs

Pulmonary diffusing capacities (DL) of NO and CO were determined simultaneously from rebreathing equilibration kinetics in anesthetized paralyzed supine dogs (mean body wt 20 kg) after denitrogenation (replacement of N2 by Ar). During rebreathing the dogs were ventilated in closed circuit with a gas mixture containing 0.06% NO, 0.06% 13C18O, and 1% He in Ar for 15 s, with tidal volume of 0.5 liter and frequency of 60/min. The partial pressures of NO, 13C18O, 16O18O, N2, Ar, CO2, and He in the trachea were continuously analyzed by mass spectrometry. Measurements were performed at various O2 levels characterized by the mean end-expired PO2 during rebreathing (PE'O2). In control conditions ("normoxia," PE'O2 = 67 +/- 8 Torr) the following mean +/- SD values were obtained (in ml.min-1.Torr-1): DLNO = 52.4 +/- 11.0 and DLCO = 15.4 +/- 2.9. In hypoxia (PE'O2 = 24 +/- 7 Torr) DLNO increased by 11 +/- 8% and DLCO by 19 +/- 10%, and in hyperoxia (PE'O2 = 390 +/- 26 Torr) DLNO decreased to 87 +/- 3% and DLCO to 56 +/- 8% with respect to values in normoxia. DLNO/DLCO of 3.24 +/- 0.06 (hypoxia), 3.38 +/- 0.31 (normoxia), and 5.54 +/- 1.04 (hyperoxia) were significantly higher than the NO/CO Krogh diffusion constant ratio (1.92) predicted for simple diffusion through aqueous layers. With increasing O2 uptake elicited by 2,4-dinitrophenol, DLNO and DLCO increased and DLNO/DLCO remained close to unchanged. The results suggest that the combined effects of diffusion and chemical reaction with hemoglobin limit alveolar-capillary transport of CO. If it is assumed that reaction kinetics of NO with hemoglobin (known to be extremely fast) are not rate limiting for NO uptake, the contribution of the slow chemical reaction with hemoglobin to the total CO uptake resistance (= 1/DLCO) was estimated to be 38% in hypoxia, 41% in normoxia, and 64% in hyperoxia. The various factors expected to restrict the validity of this analysis are discussed, in particular the effects of functional inhomogeneity.     

A "sliding scale rule" for selectivity among NO, CO, and O₂ by heme protein sensors

Selectivity among NO, CO, and O? is crucial for the physiological function of most heme proteins. Although there is a million-fold variation in equilibrium dissociation constants (K(D)), the ratios for NO:CO:O? binding stay roughly the same, 1:~10(3):~10(6), when the proximal ligand is a histidine and the distal site is apolar. For these proteins, there is a "sliding scale rule" for plots of log(K(D)) versus ligand type that allows predictions of K(D) values if one or two are missing. The predicted K(D) for binding of O?to Ns H-NOX coincides with the value determined experimentally at high pressures. Active site hydrogen bond donors break the rule and selectively increase O? affinity with little effect on CO and NO binding. Strong field proximal ligands such as thiolate, tyrosinate, and imidazolate exert a "leveling" effect on ligand binding affinity. The reported picomolar K(D) for binding of NO to sGC deviates even more dramatically from the sliding scale rule, showing a NO:CO K(D) ratio of 1:~10(8). This deviation is explained by a complex, multistep process, in which an initial low-affinity hexacoordinate NO complex with a measured K(D) of ≈54 nM, matching that predicted from the sliding scale rule, is formed initially and then is converted to a high-affinity pentacoordinate complex. This multistep six-coordinate to five-coordinate mechanism appears to be common to all NO sensors that exclude O? binding to capture a lower level of cellular NO and prevent its consumption by dioxygenation.     

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.