Distal Heme Pocket Research Articles

Carbon Monoxide Recombination Dynamics in Truncated Hemoglobins Studied with Visible-Pump MidIR-Probe Spectroscopy

Carbon monoxide recombination dynamics upon photodissociation with visible light has been characterized by means of ultrafast visible-pump/MidIR probe spectroscopy for the truncated hemoglobins from Thermobifida fusca and Bacillus subtilis. Photodissociation has been induced by exciting the sample at two different wavelengths: 400 nm, corresponding to the heme absorption in the B-band, and 550 nm, in the Q-bands. The bleached iron-CO coordination band located at 1850-1950 cm(-1) and the free CO absorption band in the region 2050-2200 cm(-1) have been observed by probe pulses tuned in the appropriate infrared region. The kinetic traces measured at 1850-1950 cm(-1) reveal multiexponential subnanosecond dynamics that have been interpreted as arising from fast geminate recombination of the photolyzed CO. A compared analysis of the crystal structure of the two proteins reveals a similar structure of their distal heme pocket, which contains conserved polar and aromatic amino acid residues closely interacting with the iron ligand. Although fast geminate recombination is observed in both proteins, several kinetic differences can be evidenced, which can be interpreted in terms of a different structural flexibility of the corresponding heme distal pockets. The analysis of the free CO band-shape and of its dynamic evolution brings out novel features about the nature of the docking site inside the protein cavity.

Chemical Rescue of the Distal Histidine Mutants of Tryptophan 2,3-Dioxygenase

Tryptophan 2,3-dioxygenase (TDO) is a heme-dependent enzyme that catalyzes the oxidative degradation of L-tryptophan (L-Trp) to N-formylkynurenine (NFK). A highly conserved histidine residue in the distal heme pocket has attracted great attention in the mechanistic studies of TDO. However, a consensus has not been reached regarding whether and how this distal histidine plays a catalytic role after substrate binding. In this study, three mutant proteins, H72S, H72N, and Q73F were generated to investigate the function of the distal histidine residue in Cupriavidus metallidurans TDO (cmTDO). Spectroscopic characterizations, enzymatic kinetic analysis, and chemical rescue assays were employed to study the biochemical properties of the wild-type enzyme and the mutant proteins. Rapid kinetic methods were utilized to explore the molecular basis for the observed stimulation of catalytic activity by 2-methylimidazole in the His72 variants. The results indicate that the distal histidine plays multiple roles in cmTDO. First, His72 contributes to but is not essential for substrate binding. In addition, it shields the heme center from nonproductive binding of exogenous small ligand molecules (i.e., imidazole and its analogs) via steric hindrance. Most importantly, His72 participates in the subsequent chemical catalytic steps after substrate binding possibly by providing H-bonding interactions to the heme-bound oxygen.

Heterogeneous Kinetics of the Carbon Monoxide Association and Dissociation Reaction to Nitrophorin 4 and 7 Coincide with Structural Heterogeneity of the Gate-Loop

NO is an important signaling molecule in human tissue. However, the mechanisms by which this molecule is controlled and directed are currently little understood. Nitrophorins (NPs) comprise a group of ferriheme proteins originating from blood-sucking insects that are tailored to protect and deliver NO via coordination to and release from the heme iron. Therefore, the kinetics of the association and dissociation reactions were studied in this work using the ferroheme-CO complexes of NP4, NP4(D30N), and NP7 as isoelectronic models for the ferriheme-NO complexes. The kinetic measurements performed by nanosecond laser-flash-photolysis and stopped-flow are accompanied by resonance Raman and FT-IR spectroscopy to characterize the carbonyl species. Careful analysis of the CO rebinding kinetics reveals that in NP4 and, to a larger extent, NP7 internal gas binding cavities are located, which temporarily trap photodissociated ligands. Moreover, changes in the free energy barriers throughout the rebinding and release pathway upon increase of the pH are surprisingly small in case of NP4. Also in case of NP4, a heterogeneous kinetic trace is obtained at pH 7.5, which corresponds to the presence of two carbonyl species in the heme cavity that are seen in vibrational spectroscopy and that are due to the change of the distal heme pocket polarity. Quantification of the two species from FT-IR spectra allowed the fitting of the kinetic traces as two processes, corresponding to the previously reported open and closed conformation of the A-B and G-H loops. With the use of the A-B loop mutant NP4(D30N), it was confirmed that the kinetic heterogeneity is controlled by pH through the disruption of the H-bond between the Asp30 side chain and the Leu130 backbone carbonyl. Overall, this first study on the slow phase of the dynamics of diatomic gas molecule interaction with NPs comprises an important experimental contribution for the understanding of the dynamics involved in the binding/release processes of NO/CO in NPs.

An Unconventional Hexacoordinated Flavohemoglobin from Mycobacterium tuberculosis

Being an obligate aerobe, Mycobacterium tuberculosis faces a number of energetic challenges when it encounters hypoxia and environmental stress during intracellular infection. Consequently, it has evolved innovative strategies to cope with these unfavorable conditions. Here, we report a novel flavohemoglobin (MtbFHb) from M. tuberculosis that exhibits unique features within its heme and reductase domains distinct from conventional FHbs, including the absence of the characteristic hydrogen bonding interactions within the proximal heme pocket and mutations in the FAD and NADH binding regions of the reductase domain. In contrast to conventional FHbs, it has a hexacoordinate low-spin heme with a proximal histidine ligand lacking imidazolate character and a distal heme pocket with a relatively low electrostatic potential. Additionally, MtbFHb carries a new FAD binding site in its reductase domain similar to that of D-lactate dehydrogenase (D-LDH). When overexpressed in Escherichia coli or Mycobacterium smegmatis, MtbFHb remained associated with the cell membrane and exhibited D-lactate:phenazine methosulfate reductase activity and oxidized D-lactate into pyruvate by converting the heme iron from Fe(3+) to Fe(2+) in a FAD-dependent manner, indicating electron transfer from D-lactate to the heme via FAD cofactor. Under oxidative stress, MtbFHb-expressing cells exhibited growth advantage with reduced levels of lipid peroxidation. Given the fact that D-lactate is a byproduct of lipid peroxidation and that M. tuberculosis lacks the gene encoding D-LDH, we propose that the novel D-lactate metabolizing activity of MtbFHb uniquely equips M. tuberculosis to balance the stress level by protecting the cell membrane from oxidative damage via cycling between the Fe(3+)/Fe(2+) redox states.

Understanding How the Distal Environment Directs Reactivity in Chlorite Dismutase: Spectroscopy and Reactivity of Arg183 Mutants

The chlorite dismutase from Dechloromonas aromatica (DaCld) catalyzes the highly efficient decomposition of chlorite to O(2) and chloride. Spectroscopic, equilibrium thermodynamic, and kinetic measurements have indicated that Cld has two pH sensitive moieties; one is the heme, and Arg183 in the distal heme pocket has been hypothesized to be the second. This active site residue has been examined by site-directed mutagenesis to understand the roles of positive charge and hydrogen bonding in O-O bond formation. Three Cld mutants, Arg183 to Lys (R183K), Arg183 to Gln (R183Q), and Arg183 to Ala (R183A), were investigated to determine their respective contributions to the decomposition of chlorite ion, the spin state and coordination states of their ferric and ferrous forms, their cyanide and imidazole binding affinities, and their reduction potentials. UV-visible and resonance Raman spectroscopies showed that DaCld(R183A) contains five-coordinate high-spin (5cHS) heme, the DaCld(R183Q) heme is a mixture of five-coordinate and six-coordinate high spin (5c/6cHS) heme, and DaCld(R183K) contains six-coordinate low-spin (6cLS) heme. In contrast to wild-type (WT) Cld, which exhibits pK(a) values of 6.5 and 8.7, all three ferric mutants exhibited pH-independent spectroscopic signatures and kinetic behaviors. Steady state kinetic parameters of the chlorite decomposition reaction catalyzed by the mutants suggest that in WT DaCld the pK(a) of 6.5 corresponds to a change in the availability of positive charge from the guanidinium group of Arg183 to the heme site. This could be due to either direct acid-base chemistry at the Arg183 side chain or a flexible Arg183 side chain that can access various orientations. Current evidence is most consistent with a conformational adjustment of Arg183. A properly oriented Arg183 is critical for the stabilization of anions in the distal pocket and for efficient catalysis.

Proline 107 Is a Major Determinant in Maintaining the Structure of the Distal Pocket and Reactivity of the High-Spin Heme of MauG

The diheme enzyme MauG catalyzes a six-electron oxidation required for posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. Crystallographic studies had shown that Pro107, which resides in the distal pocket of the high-spin heme of MauG, changes conformation upon binding of CO or NO to the heme iron. In this study, Pro107 was converted to Cys, Val, and Ser by site-directed mutagenesis. The structures of each of these MauG mutant proteins in complex with preMADH were determined, as were their physical and catalytic properties. P107C MauG was inactive, and the crystal structure revealed that Cys107 had been oxidatively modified to a sulfinic acid. Mass spectrometry revealed that this modification was present prior to crystallization. P107V MauG exhibited spectroscopic and catalytic properties that were similar to those of wild-type MauG, but P107V MauG was more susceptible to oxidative damage. The P107S mutation caused a structural change that resulted in the five-coordinate high-spin heme being converted to a six-coordinate heme with a distal axial ligand provided by Glu113. EPR and resonance Raman spectroscopy revealed this heme remained high-spin but with greatly increased rhombicity as compared to that of the axial signal of wild-type MauG. P107S MauG was resistant to reduction by dithionite and reaction with H(2)O(2) and unable to catalyze TTQ biosynthesis. These results show that the presence of Pro107 is critical in maintaining the proper structure of the distal heme pocket of the high-spin heme of MauG, allowing exogenous ligands to bind and directing the reactivity of the heme-activated oxygen during catalysis, thus minimizing the oxidation of other residues of MauG.

Study on specific interaction of new ferrocene-substituted carborane conjugates with hemoglobin protein

The interactions between the new organometallic complexes, ferrocene-substituted dithio-o-carborane conjugates (denoted as FcSB1, FcSB2 and FcSBCO) and hemoglobin (Hb) are investigated by electrochemistry, fluorescence and UV-vis absorption spectroscopy. The results demonstrate that FcSB1, FcSB2 and FcSBCO can bind to the heme iron center through the replacement of the weakly bound H2O/O2 in the distal heme pocket of Hb by their sulfur donor atoms, inducing the allosteric change from the R state (oxygenated conformation, relax) to T state (deoxygenated conformation, tense). The binding affinity is in the order of FcSBCO>FcSB2>FcSB1. Moreover, the fluorescence study illustrates that the three ferrocene-carborane conjugates differently affect the quarterly and tertiary structures as well as the polarity in the surrounding of the Trp and Tyr residues in Hb. Typically, FcSB2 mainly induces alterations of the microenvironment around the β37Trp residue which is located on the α1β2 interface of Hb. Such distinct influences are attributed to the structural features of FcSB1, FcSB2 and FcSBCO containing hydrophobic ferrocenyl and carboranyl units as well as C=O group. Screening the protein-binding behavior can signify the potential bioactivity of such molecules and may be helpful in the future development of promising multifunctional metallodrugs.

Structure and Dynamics of Mycobacterium tuberculosis Truncated Hemoglobin N: Insights from NMR Spectroscopy and Molecular Dynamics Simulations

The potent nitric oxide dioxygenase (NOD) activity (trHbN-Fe²⁺-O₂ + (•)NO → trHbN-Fe³⁺-OH₂ + NO₃⁻) of Mycobacterium tuberculosis truncated hemoglobin N (trHbN) protects aerobic respiration from inhibition by (•)NO. The high activity of trHbN has been attributed in part to the presence of numerous short-lived hydrophobic cavities that allow partition and diffusion of the gaseous substrates (•)NO and O₂ to the active site. We investigated the relation between these cavities and the dynamics of the protein using solution NMR spectroscopy and molecular dynamics (MD). Results from both approaches indicate that the protein is mainly rigid with very limited motions of the backbone N-H bond vectors on the picoseconds-nanoseconds time scale, indicating that substrate diffusion and partition within trHbN may be controlled by side-chains movements. Model-free analysis also revealed the presence of slow motions (microseconds-milliseconds), not observed in MD simulations, for many residues located in helices B and G including the distal heme pocket Tyr33(B10). All currently known crystal structures and molecular dynamics data of truncated hemoglobins with the so-called pre-A N-terminal extension suggest a stable α-helical conformation that extends in solution. Moreover, a recent study attributed a crucial role to the pre-A helix for NOD activity. However, solution NMR data clearly show that in near-physiological conditions these residues do not adopt an α-helical conformation and are significantly disordered and that the helical conformation seen in crystal structures is likely induced by crystal contacts. Although this lack of order for the pre-A does not disagree with an important functional role for these residues, our data show that one should not assume an helical conformation for these residues in any functional interpretation. Moreover, future molecular dynamics simulations should not use an initial α-helical conformation for these residues in order to avoid a bias based on an erroneous initial structure for the N-termini residues. This work constitutes the first study of a truncated hemoglobin dynamics performed by solution heteronuclear relaxation NMR spectroscopy.

Strong Ligand–Protein Interactions Revealed by Ultrafast Infrared Spectroscopy of CO in the Heme Pocket of the Oxygen Sensor FixL

In heme-based sensor proteins, ligand binding to heme in a sensor domain induces conformational changes that eventually lead to changes in enzymatic activity of an associated catalytic domain. The bacterial oxygen sensor FixL is the best-studied example of these proteins and displays marked differences in dynamic behavior with respect to model globin proteins. We report a mid-IR study of the configuration and ultrafast dynamics of CO in the distal heme pocket site of the sensor PAS domain FixLH, employing a recently developed method that provides a unique combination of high spectral resolution and range and high sensitivity. Anisotropy measurements indicate that CO rotates toward the heme plane upon dissociation, as is the case in globins. Remarkably, CO bound to the heme iron is tilted by ~30° with respect to the heme normal, which contrasts to the situation in myoglobin and in present FixLH-CO X-ray crystal structure models. This implies protein-environment-induced strain on the ligand, which is possibly at the origin of a very rapid docking-site population in a single conformation. Our observations likely explain the unusually low affinity of FixL for CO that is at the origin of the weak ligand discrimination between CO and O(2). Moreover, we observe orders of magnitude faster vibrational relaxation of dissociated CO in FixL than in globins, implying strong interactions of the ligand with the distal heme pocket environment. Finally, in the R220H FixLH mutant protein, where CO is H-bonded to a distal histidine, we demonstrate that the H-bond is maintained during photolysis. Comparison with extensively studied globin proteins unveils a surprisingly rich variety in both structural and dynamic properties of the interaction of a diatomic ligand with the ubiquitous b-type heme-proximal histidine system in different distal pockets.

The optical spectra of fluoride complexes can effectively probe H-bonding interactions in the distal cavity of heme proteins

Fluoride complexes of heme proteins are characterized by unique spectroscopic properties, that provide a simple and direct means to monitor the interactions of the distal heme pocket environment with the iron-bound ligand. In particular, a strong correlation has been demonstrated between the wavelength of the iron-porphyrin charge transfer band at 600–620 nm (CT1) and the strength of H-bonding donation from the distal amino acid side chains to the fluoride ion. In parallel, resonance Raman spectra with excitation within either the CT1 band or the charge transfer band at 450–460 nm (CT2) have revealed that the iron-fluoride stretching frequency is directly affected by H-bonding to the fluoride ion. On this basis, globins and peroxidases display distinct spectroscopic features, which are strongly dependent on the capability of their distal residues (i.e. histidine, arginine and tryptophan) to be involved in H-bonding with the ligand. In particular, in peroxidases strong H-bonding corresponds to a low iron-fluoride stretching frequency and to a red-shifted CT1 band. The reverse is observed in myoglobin. Interestingly, a truncated hemoglobin of microbial origin ( Thermobifida fusca) investigated in the present work, displays the specific spectroscopic signature of a peroxidase, in agreement with the presence of strong H-bonding residues, i.e., tyrosine and tryptophan, within the distal pocket.

Electronic properties of the highly ruffled heme bound to the heme degrading enzyme IsdI

IsdI, a heme-degrading protein from Staphylococcus aureus, binds heme in a manner that distorts the normally planar heme prosthetic group to an extent greater than that observed so far for any other heme-binding protein. To understand better the relationship between this distinct structural characteristic and the functional properties of IsdI, spectroscopic, electrochemical, and crystallographic results are reported that provide evidence that this heme ruffling is essential to the catalytic activity of the protein and eliminates the need for the water cluster in the distal heme pocket that is essential for the activity of classical heme oxygenases. The lack of heme orientational disorder in (1)H-NMR spectra of the protein argues that the catalytic formation of β- and δ-biliverdin in nearly equal yield results from the ability of the protein to attack opposite sides of the heme ring rather than from binding of the heme substrate in two alternative orientations.

The Proximal Hydrogen Bond Network Modulates Bacillus subtilis Nitric-oxide Synthase Electronic and Structural Properties

Bacterial nitric-oxide synthase (NOS)-like proteins are believed to be genuine NOSs. As for cytochromes P450 (CYPs), NOS-proximal ligand is a thiolate that exerts a push effect crucial for the process of dioxygen activation. Unlike CYPs, this catalytic electron donation seems controlled by a hydrogen bond (H-bond) interaction between the thiolate ligand and a vicinal tryptophan. Variations of the strength of this H-bond could provide a direct way to tune the stability along with the electronic and structural properties of NOS. We generated five different mutations of bsNOS Trp66, which can modulate this proximal H-bond. We investigated the effects of these mutations on different NOS complexes (FeIII, FeIICO, and FeIINO), using a combination of UV-visible absorption, EPR, FTIR, and resonance Raman spectroscopies. Our results indicate that (i) the proximal H-bond modulation can selectively decrease or increase the electron donating properties of the proximal thiolate, (ii) this modulation controls the σ-competition between distal and proximal ligands, (iii) this H-bond controls the stability of various NOS intermediates, and (iv) a fine tuning of the electron donation by the proximal ligand is required to allow at the same time oxygen activation and to prevent uncoupling reactions.

Histidine E7 Dynamics Modulates Ligand Exchange between Distal Pocket and Solvent in AHb1 from Arabidopsis thaliana

The distal His residue in type 1 nonsymbiotic hemoglobin AHb1 from Arabidopsis thaliana plays a fundamental role in stabilizing the bound ligand. This residue might also be important in regulating the accessibility to the distal cavity. The feasibility of this functional role has been examined using a combination of experimental and computational methods. We show that the exchange of CO between the solvent and the reaction site is modulated by a swinging motion of the distal His, which opens a channel that connects directly the distal heme pocket with the solvent. The nearby PheB10 aids the distal His in the stabilization of the bound ligand by providing additional protection against solvation. Overall, these findings provide evidence supporting the functional implications of the conformational rearrangement found for the distal His in AHb1, which mimics the gating role proposed for the same residue in myoglobin.

Crystal Structures of CO and NO Adducts of MauG in Complex with Pre-Methylamine Dehydrogenase: Implications for the Mechanism of Dioxygen Activation

MauG is a diheme enzyme responsible for the post-translational formation of the catalytic tryptophan tryptophylquinone (TTQ) cofactor in methylamine dehydrogenase (MADH). MauG can utilize hydrogen peroxide, or molecular oxygen and reducing equivalents, to complete this reaction via a catalytic bis-Fe(IV) intermediate. Crystal structures of diferrous, Fe(II)-CO, and Fe(II)-NO forms of MauG in complex with its preMADH substrate have been determined and compared to one another as well as to the structure of the resting diferric MauG-preMADH complex. CO and NO each bind exclusively to the 5-coordinate high-spin heme with no change in ligation of the 6-coordinate low-spin heme. These structures reveal likely roles for amino acid residues in the distal pocket of the high-spin heme in oxygen binding and activation. Glu113 is implicated in the protonation of heme-bound diatomic oxygen intermediates in promoting cleavage of the O-O bond. Pro107 is shown to change conformation on the binding of each ligand and may play a steric role in oxygen activation by positioning the distal oxygen near Glu113. Gln103 is in a position to provide a hydrogen bond to the Fe(IV)═O moiety that may account for the unusual stability of this species in MauG.

Probing the local electronic and geometric properties of the heme iron center in a H-NOX domain

Heme-Nitric oxide and/or OXygen binding (H-NOX) proteins are a family of diatomic gas binding hemoproteins that have attracted intense research interest. Here we employ X-ray absorption near-edge structure (XANES) spectroscopy to study the nitric oxide (NO) binding site of H-NOX. This is the first time this technique has been utilized to examine the NO/H-NOX signaling pathway. XANES spectra of wildtype and a point mutant (proline 115 to alanine, P115A) of the H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX) were obtained and analyzed for ferrous and ferric complexes of the protein. This work provides specific structural characterization of the solution state of several Tt H-NOX ferrous complexes (− unligated, − NO, and − CO) that were previously unavailable. Our iron K-edges indicate effective charge on the iron center in the various complexes and report on the electronic environment of heme iron. We analyzed the ligand field indicator ratio (LFIR), which is extracted from XANES spectra, for each complex, providing an understanding of ligand field strength, spin state of the central iron, movement of the iron atom upon ligation, and ligand binding properties. In particular, our LFIRs indicate that the heme iron is dramatically displaced towards the distal pocket during ligand binding. Based on these results, we propose that iron displacement towards the distal heme pocket is an essential step in signal initiation in H-NOX proteins. This provides a mechanistic link between ligand binding and the changes in heme and protein conformation that have been observed for H-NOX family members during signaling.

Ligand migration in the truncated hemoglobin of Mycobacterium tuberculosis

The truncated hemoglobin of Mycobacterium tuberculosis (Mt-trHbO) is a small heme protein belonging to the hemoglobin superfamily. Truncated hemoglobins (trHbs) are believed to have functional roles such as terminal oxidases and oxygen sensors involved in the response to oxidative and nitrosative stress, nitric oxide (NO) detoxification, O₂/NO chemistry, O₂ delivery under hypoxic conditions, and long-term ligand storage. Based on sequence similarities, they are classified into three groups. Experimental studies revealed that all trHbs display a 2-on-2 α-helical sandwich fold rather than the 3-on-3 α-helical sandwich fold of the classical hemoglobin fold. Using locally enhanced sampling (LESMD) molecular dynamics, the ligand-binding escape pathways from the distal heme binding cavity of Mt-trHbO were determined to better understand how this protein functions. The importance of specific residues, such as the group II and III invariant W(G8) residue, can be seen in terms of ligand diffusion pathways and ligand dynamics. LESMD simulations show that the wild-type Mt-trHbO has three diffusion pathways while the W(G8)F Mt-trHbO mutant has only two. The W(G8) residue plays a critical role in ligand binding and stabilization and helps regulate the rate of ligand escape from the distal heme pocket. Thus, this invariant residue is important in creating ligand diffusion pathways and possibly in the enzymatic functions of this protein.

Effect of alternative distal residues on the reactivity of cytochrome c peroxidase: Properties of CcP mutants H52D, H52E, H52N, and H52Q

To test the effect of alternative bases at the distal histidine position, four CcP variants have been constructed that substitute the two basic residues, aspartate and glutamate, and their amides, asparagine and glutamine, for histidine-52, i.e., CcP(H52D), CcP(H52E), CcP(H52N), and CcP(H52Q). All four mutants catalyze oxidation of ferrocytochrome c by H2O2 with steady-state activities that are between 250 and 7700 times slower than wild-type CcP at pH 6.0, 0.10M ionic strength, 25°C. The rate of Compound I formation is decreased between 3.5 and 5.4 orders of magnitude for the mutants compared to wild-type CcP, with the rate of the reaction between CcP(H52Q) and H2O2 the slowest yet observed for any CcP mutant. A correlation between the rate of Compound I formation and the rate of HCN binding for CcP and various CcP distal pocket mutants provides strong evidence that the rate-limiting step in CcP Compound I formation is deprotonation of H2O2 within the distal heme pocket under the experimental conditions employed in this study. While CcP(H52E) reacts stoichiometrically with H2O2 to form Compound I, only ~36% of CcP(H52D), ~21% of CcP(H52Q) and ~8% of CcP(H52N) appear to be converted to Compound I during their respective reactions with H2O2. This is partially due to the slow rate of Compound I formation and the rapid endogenous decay of Compound I for these mutants. The pathways for the endogenous decay of Compound I for the four mutants used in this study are distinct from that of wild-type CcP Compound I.

An Investigation of the Distal Histidyl H-Bonds in Oxyhemoglobin: Effects of Temperature, pH, and Inositol Hexaphosphate

Ligand binding to hemoglobin (Hb) is a dynamic process involving many tertiary and quaternary structural changes. It has been inferred that the O2 binding to Hb is stabilized by the H-bonds between the O2 ligands and the side-chains of two distal histidyl residues, α58His and β63His. Our previous study by multinuclear nuclear magnetic resonance (NMR) spectroscopy has provided the first direct evidence of such H-bonds in human normal adult oxyhemoglobin (HbO2 A) in solution. Here, the NMR spectra of uniformly 15N-labeled recombinant human Hb A (rHb A) and five mutant rHbs in the oxy form have been studied under various experimental conditions of pH, temperature, and inositol hexaphosphate (IHP). We have found that these H-bonds can be used as markers to reflect structural changes in the distal heme pocket. At lower pH and/or higher temperature, the side-chains of the distal histidines appear to be more mobile and the exchange with water molecules in the distal heme pockets is faster, which is consistent with the changes in the O2 affinity of Hb as a function of pH and temperature. Our NMR results have also confirmed that the H-bond in the β-chain is weaker than that in the α-chain and is more sensitive to changes in pH and temperature. These H-bonds are sensitive to the mutation in the distal heme pockets, but only affected indirectly by the mutations in the quaternary interfaces and IHP. The recent findings are consistent with our previous NMR results showing that IHP exerts its effects on both the tertiary and quaternary structures of Hb A. (Supported by NIH grant R01GM084614)

Structure of Ralstonia eutropha Flavohemoglobin in Complex with Three Antibiotic Azole Compounds

Flavohemoglobins (flavoHbs) are enzymes that operate primarily as nitric oxide dioxygenases and shuttle thereby electrons among NAD(P)H, FAD, heme, and a ligated redox-active substrate such as O(2). They function in the bacterial defense against nitrosative stress and are therefore considered as targets for new antibiotic drugs. Recently, azole derivatives were proven to be attractive nitric oxide dioxygenase inhibitors, and to explore their binding characteristics, we determined the X-ray structure of the flavoHb from Ralstonia eutropha in a complex with miconazole (FHP(M)), econazole (FHP(E)), and ketoconazole (FHP(K)). In agreement with UV-vis spectroscopic data, one azole compound binds inside the distal heme pocket and ligates to the heme iron by its imidazole substituent. The two additional substituents, mostly chlorinated phenyl groups, form a series of van der Waals contacts with the protein matrix. Both interactions explain their high affinity for flavoHbs, the binding constants being 2.6, 1.2, and 11.6 μM for miconazole, econazole, and ketoconazole, respectively. The FHP(M) and FHP(Lip) (flavoHbs originally loaded with a phospholipid) structures share an "open" state and the FHP(E) and FHP(K) structures a "closed" state. Although the azole compounds were able to push the lipid out of its binding site, a fatty acid fragment is still bound inside the heme pocket of FHP(E) and FHP(K) and dictates the state of the protein. The ligand-induced open-to-closed transition involves a reorientation of the NADH domain accompanied by conformational changes in the C-terminal arm, helix E, and the CE loop resulting in an encapsulation of the heme-binding pocket. Implications of the observed open-to-closed process on the catalytic cycle are discussed.

Crystal structure of the carbon monoxide complex of human cytoglobin

Cytoglobin (Cgb) is a vertebrate heme-containing globin-protein expressed in a broad range of mammalian tissues. Unlike myoglobin, Cgb displays a hexa-coordinated (bis-hystidyl) heme iron atom, having the heme distal His81(E7) residue as the endogenous sixth ligand. In the present study, we crystallized human Cgb in the presence of a reductant Na₂S₂O₄ under a carbon monoxide (CO) atmosphere, and determined the crystal structure at 2.6 A resolution. The CO ligand occupies the sixth axial position of the heme ferrous iron. Eventually, the imidazole group of His81(E7) is expelled from the sixth position and swings out of the distal heme pocket. The flipping motion of the His81 imidazole group accompanies structural readjustments of some residues (Gln62, Phe63, Gln72, and Ser75) in both the CD-corner and D-helix regions of Cgb. On the other hand, no significant structural changes were observed in other Cgb regions, for example, on the proximal side. These structural alterations that occurred as a result of exogenous ligand (CO) binding are clearly different from those observed in other vertebrate hexa-coordinated globins (mouse neuroglobin, Drosophila melanogaster hemoglobin) and penta-coordinated sperm whale myoglobin. The present study provides the structural basis for further discussion of the unique ligand-binding properties of Cgb.