The site of substrate and fructose 2,6-bisphosphate binding to rabbit liver fructose-1,6-bisphosphatase

The binding site(s) in rabbit liver fructose-1,6-bisphosphatase for the active site binding ligand, fructose 6-phosphate, and the inhibitor, fructose 2,6-bisphosphate, have been investigated by using nuclear magnetic resonance spectroscopy. The distance from a nitroxide spin label to the bound ligands and the distance from the structural metal site to the bound ligands are about the same within experimental error. These data indicate that the two ligands probably bind at the active site in the rabbit liver enzyme.     

Carbon monoxide binding to the ferrous chains of [Mn,Fe(II)] hybrid hemoglobins: pH dependence of the chain affinity constants associated with specific hemoglobin ligation pathways

In mixed-metal [Mn,Fe] hybrid hemoglobins (Hb), the two chains of a single type, alpha or beta, are substituted with manganese protoporphyrin IX, which does not bind CO in either the Mn(II) or Mn(III) valency states. Thus, CO binding by the two ferrous subunits of a hybrid with Mn of either valency represents a simplified two-step Hb ligation process in which ligands bind to a single-chain type. Considering the [Mn(II),Fe(II)] hybrids, which are deoxy T-state analogues, at pH 6.6 both types bind CO with low affinity (alpha-Fe, 0.38 mmHg; beta-Fe, 0.71 mmHg) and noncooperatively (Hill coefficient n = 1). At elevated pH, both exhibit an increase in affinity (Bohr effect) and strong cooperativity, with the alpha-Fe hybrid having a higher degree of cooperativity (n approximately equal to 1.6) than beta-Fe (approximately equal to 1.3) at pH 9.0. The CO association constants for the Hb ligation routes in which the first two ligands bind to the same chain type are obtained from these measurements, and their pH dependence provides estimates of the proton release at each step. Through studies of CO on- and off-rates, the [Mn(III),Fe(II)] hybrids are used to obtain the pH dependence of the association constants for binding the fourth CO to the individual Hb chains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complexes of horseradish peroxidase with formate, acetate, and carbon monoxide

Carbon monoxide, formate, and acetate interact with horseradish peroxidase (HRP) by binding to subsites within the active site. These ligands also bind to catalases, but their interactions are different in the two types of enzymes. Formate (notionally the "hydrated" form of carbon monoxide) is oxidized to carbon dioxide by compound I in catalase, while no such reaction is reported to occur in HRP, and the CO complex of ferrocatalase can only be obtained indirectly. Here we describe high-resolution crystal structures for HRP in its complexes with carbon monoxide and with formate, and compare these with the previously determined HRP-acetate structure [Berglund, G. I., et al. (2002) Nature 417, 463-468]. A multicrystal X-ray data collection strategy preserved the correct oxidation state of the iron during the experiments. Absorption spectra of the crystals and electron paramagnetic resonance data for the acetate and formate complexes in solution correlate electronic states with the structural results. Formate in ferric HRP and CO in ferrous HRP bind directly to the heme iron with iron-ligand distances of 2.3 and 1.8 A, respectively. CO does not bind to the ferric iron in the crystal. Acetate bound to ferric HRP stacks parallel with the heme plane with its carboxylate group 3.6 A from the heme iron, and without an intervening solvent molecule between the iron and acetate. The positions of the oxygen atoms in the bound ligands outline a potential access route for hydrogen peroxide to the iron. We propose that interactions in this channel ensure deprotonation of the proximal oxygen before binding to the heme iron.     

Influence of Distal Residue B10 on CO Dynamics in Myoglobin and Neuroglobin

For many years, myoglobin has served as a paradigm for structure–function studies in proteins. Ligand binding and migration within myoglobin has been studied in great detail by crystallography and spectroscopy, showing that gaseous ligands such as O₂, CO, and NO not only bind to the heme iron but may also reside transiently in three internal ligand docking sites, the primary docking site B and secondary sites C and D. These sites affect ligand association and dissociation in specific ways. Neuroglobin is another vertebrate heme protein that also binds small ligands. Ligand migration pathways in neuroglobin have not yet been elucidated. Here, we have used Fourier transform infrared temperature derivative spectroscopy at cryogenic temperatures to compare the influence of the side chain volume of amino acid residue B10 on ligand migration to and rebinding from docking sites in myoglobin and neuroglobin.     

Aryl cyclopentadienyl tricarbonyl rhenium complexes: novel ligands for the estrogen receptor with potential use as estrogen radiopharmaceuticals

The need for imaging agents for estrogen receptor positive (ER+) tumors that are both cost effective and widely available, as well as the need for novel radiotherapeutic agents for the treatment of breast cancer, has prompted us to investigate cyclopentadienyl tricarbonyl metal [CpMet(CO)(3), Met=Re, Tc-99m] complexes that bind well to the ER. Thus, we have prepared a series of p-hydroxyphenyl-substituted CpRe(CO)(3) complexes and evaluated them (and, in some cases, their cyclopentadiene precursors) for binding to ER. These compounds constitute a new class of structurally integrated organometallic ligands for ER in which the CpMet(CO)(3 )organometallic unit forms the very structural core of these molecules and thus is necessarily intimately involved in their interaction with the receptor. The CpRe(CO)(3) compounds were prepared by reaction of the lithium salt of the arene-substituted cyclopentadiene with a suitable Re(CO)(3)(+) precursor, followed by deprotection of the methyl ether. The X-ray crystal structure of one of these analogues shows that it has the classical 'piano stool'-like geometry, with the alkyl groups directed upward, away from the tripodyl metal carbonyl base. The aryl-substituted CpRe(CO)(3) complexes that we have prepared all bind to the ER, some with affinity as great as 20% that of the native ligand, estradiol. In general, at least two p-hydroxyphenyl substituents and one to two alkyl groups attached to the organometallic cyclopentadienyl core are needed for high ER affinity. Where we have been able to make comparisons, the metal complexes bind to ER with an affinity greater than their cyclopentadiene precursors. The high affinity of some of these complexes indicates that the bulky Re(CO)(3) unit is able to exploit the considerable volume in the center of the ER ligand binding pocket that is not occupied by most ligands, a consideration that is supported by molecular modeling. The preparation of the best of these agents in technetium-99m labeled form is currently being investigated.     

The one-electron autoxidation of human cytochrome P450 3A4

Monomeric cytochrome P450 3A4 (CYP3A4), the most prevalent cytochrome P450 in human liver, can simultaneously bind one, two, or three molecules of substrates and effectors. The difference in the functional properties of such binding intermediates gives rise to homotropic and heterotropic cooperative kinetics of this enzyme. To understand the overall kinetic processes operating in CYP3A4, we documented the kinetics of autoxidation of the oxy-ferrous intermediate of CYP3A4 as a function of testosterone concentration. The rate of autoxidation in the presence of testosterone was significantly lower than that observed with no substrate present. Stability of the oxy-ferrous complex in CYP3A4 and the amplitude of the geminate CO rebinding increased significantly as a result of binding of just one testosterone molecule. In contrast, the slow phase in the kinetics of cyanide binding to the ferric CYP3A4 correlated with a shift of the heme iron spin state, which is only caused by the association of a second molecule of testosterone. Our results show that the first substrate binding event prevents the escape of diatomic ligands from the distal heme binding pocket, stabilizes the oxy-ferrous complex, and thus serves as an important modulator of the uncoupling channel in the cytochromes P450.     

Quadruplex ligands may act as molecular chaperones for tetramolecular quadruplex formation

G-quadruplexes are a family of four-stranded DNA structures, stabilized by G-quartets, that form in the presence of monovalent cations. Efforts are currently being made to identify ligands that selectively bind to G-quadruplex motifs as these compounds may interfere with the telomere structure, telomere elongation/replication and proliferation of cancer cells. The kinetics of quadruplex–ligands interactions are poorly understood: it is not clear whether quadruplex ligands lock into the preformed structure (i.e. increase the lifetime of the structure by lowering the dissociation constant, k_off) or whether ligands actively promote the formation of the complex and act as quadruplex chaperones by increasing the association constant, k_on. We studied the effect of a selective quadruplex ligand, a bisquinolinium pyridine dicarboxamide compound called 360A, to distinguish these two possibilities. We demonstrated that, in addition to binding to and locking into preformed quadruplexes, this molecule acted as a chaperone for tetramolecular complexes by acting on k_on. This observation has implications for in vitro and in vivo applications of quadruplexes and should be taken into account when evaluating the cellular responses to these agents.     

Interaction of cytochrome bd with carbon monoxide at low and room temperatures: evidence that only a small fraction of heme b595 reacts with CO

Azotobacter vinelandii is an obligately aerobic bacterium in which aerotolerant dinitrogen fixation requires cytochrome bd. This oxidase comprises two polypeptide subunits and three hemes, but no copper, and has been studied extensively. However, there remain apparently conflicting reports on the reactivity of the high spin heme b(595) with ligands. Using purified cytochrome bd, we show that absorption changes induced by CO photodissociation from the fully reduced cytochrome bd at low temperatures demonstrate binding of the ligand with heme b(595). However, the magnitude of these changes corresponds to the reaction with CO of only about 5% of the heme. CO binding with a minor fraction of heme b(595) is also revealed at room temperature by time-resolved studies of CO recombination. The data resolve the apparent discrepancies between conclusions drawn from room and low temperature spectroscopic studies of the CO reaction with cytochrome bd. The results are consistent with the proposal that hemes b(595) and d form a diheme oxygen-reducing center with a binding capacity for a single exogenous ligand molecule that partitions between the hemes d and b(595) in accordance with their intrinsic affinities for the ligand. In this model, the affinity of heme b(595) for CO is about 20-fold lower than that of heme d.     

Stathmin and Interfacial Microtubule Inhibitors Recognize a Naturally Curved Conformation of Tubulin Dimers

Tubulin is able to switch between a straight microtubule-like structure and a curved structure in complex with the stathmin-like domain of the RB3 protein (T₂RB3). GTP hydrolysis following microtubule assembly induces protofilament curvature and disassembly. The conformation of the labile tubulin heterodimers is unknown. One important question is whether free GDP-tubulin dimers are straightened by GTP binding or if GTP-tubulin is also curved and switches into a straight conformation upon assembly. We have obtained insight into the bending flexibility of tubulin by analyzing the interplay of tubulin-stathmin association with the binding of several small molecule inhibitors to the colchicine domain at the tubulin intradimer interface, combining structural and biochemical approaches. The crystal structures of T₂RB3 complexes with the chiral R and S isomers of ethyl-5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl-carbamate, show that their binding site overlaps with colchicine ring A and that both complexes have the same curvature as unliganded T₂RB3. The binding of these ligands is incompatible with a straight tubulin structure in microtubules. Analytical ultracentrifugation and binding measurements show that tubulin-stathmin associations (T₂RB3, T₂Stath) and binding of ligands (R, S, TN-16, or the colchicine analogue MTC) are thermodynamically independent from one another, irrespective of tubulin being bound to GTP or GDP. The fact that the interfacial ligands bind equally well to tubulin dimers or stathmin complexes supports a bent conformation of the free tubulin dimers. It is tempting to speculate that stathmin evolved to recognize curved structures in unassembled and disassembling tubulin, thus regulating microtubule assembly.     

The reactions of neuroglobin with CO: evidence for two forms of the ferrous protein

The normally hexa coordinate ferrous form of neuroglobin binds CO by replacement of the heme-linked distal histidine residue. We have studied this reaction in detail using stopped flow techniques. The reaction time courses are complex at all the wavelengths studied. Specifically the reaction with CO occurs in two temporally separable phases, each of which shows a hyperbolic dependence of rate on CO concentration, indicating they each arise from histidine replacement by CO. Analysis of the observed rates as a function of the CO concentration, measured in the pH range 6.0-8.0, allows us to determine both the rate of histidine-heme ligand binding and dissociation for each of the two forms of the protein present in solution at each pH value. The pH dependence of the histidine association and dissociation rates is complex, as are the derived equilibrium constants for distal histidine binding. The spectral change associated with each reaction phase is very similar and independent of the CO concentration, showing that the two protein forms responsible for the two observed kinetic processes are not in equilibrium on the time scale of our investigations. Our data suggests that, unlike many other heme proteins, neuroglobin displays complex reactivity with ligands in the ferrous form due to heme rotational disorder, as has previously been reported for the ferric form of the protein.     

Infrared Study of Carbon Monoxide Migration among Internal Cavities of Myoglobin Mutant L29W

Myoglobin, a small globular heme protein that binds gaseous ligands such asO₂, CO and NO reversibly at the heme iron, provides an excellent modelsystem for studying structural and dynamic aspects of protein reactions. Flashphotolysis experiments, performed over wide ranges in time and temperature, reveal a complex ligand binding reaction with multiple kinetic intermediates, resulting from protein relaxation and movements of the ligand within the protein. Our recent studies of carbonmonoxy-myoglobin (MbCO) mutant L29W, using time-resolved infrared spectroscopy in combination with x-ray crystallography, have correlated kinetic intermediates with photoproduct structures that are characterized by the CO residing in different internal protein cavities, so-called xenon holes. Here we have used Fourier transform infrared temperature derivative spectroscopy (FTIR-TDS) to further examine the role of internal cavities in the dynamics. Different cavities can be accessed by the CO ligands at different temperatures, and characteristic infrared absorption spectra have been obtained for the different locations of the CO ligand within the protein, enabling us to monitor ligand migration through the protein as well as conformational changes of the protein.     

Use of the multiple copy simultaneous search (MCSS) method to design a new class of picornavirus capsid binding drugs

A combinatorial ligand design approach based on the multiple copy simultaneous search (MCSS) method and a simple scheme for joining MCSS functional group sites was applied to the binding pocket of P3/Sabin poliovirus and rhinovirus 14. The MCSS method determines where specific functional (chemical) groups have local potential energy minima in the binding site. Before the virus application, test calculations were run to determine the optimal set of input parameters to be used in evaluating the MCSS results. The MCSS minima are analyzed and selected minima are connected with (CH₂) _n linkers to form candidate ligands, whose structures are optimized in the binding site. Estimates of the binding strength were made for the ligands and compared with those for known drugs. The results indicate that the proposed ligands should bind to P3/Sabin poliovirus at least as well as the best of the existing drugs, and that they should also bind to P1/Mahoney poliovirus and rhinovirus 14. A detailed comparison of the poliovirus and rhinovirus binding pockets and an analysis of drug binding specificity is presented. Proteins 29:32–58, 1997. © 1997 Wiley-Liss, Inc.     

The role of entropy in the discrimination between CO and O2 in myoglobin

Using stopped-flow rapid mixing and flash photolysis techniques, the dissociation rate coefficients of horse carbonmonoxy myoglobin (hMbCO) and oxygenated myoglobin (hMbO₂) in aqueous solution have been determined as a function of temperature between 274 and 342 K. From the Arrhenius plot, an activation enthalpy for dissociation of 74 kJ/mol was obtained for both ligands. The pronounced kinetic differences arise from markedly different pre-exponentials. We compare the Arrhenius parameters with those of the association reaction, as measured at cryogenic temperatures. In our analysis we conclude that the entropy loss upon binding of O₂ is twice as large as that for CO. Taking reasonable estimates for the frequency factor, the transition state entropy in hMbO₂ is located roughly half way in between the entropies of the bound and unbound states. By contrast, the entropy of the transition state in hMbCO appears to be identical to that of the bound state. Possible structural reasons for the different behavior are discussed. Received: 13 January 1997 / Accepted: 24 April 1997     

Spectroscopic and kinetic features of allocolchicine binding to tubulin

Allocolchicine is a structural isomer of colchicine in which colchicine's tropone C ring is replaced with an aromatic ester. In spite of the structural differences between the two ligands, the association parameters for both molecules binding to tubulin are quite similar. The association constant for allocolchicine binding to tubulin was determined by fluorescence titration to be 6.1 x 10(5) M-1 at 37 degrees C, which is about a factor of 5 less than that of the colchicine-tubulin association. In particular, analysis of the kinetics of the association of allocolchicine with tubulin yielded nearly equivalent activation parameters for the two ligands. The activation energy of the allocolchicine binding reaction was found to be 18.4 +/- 1.5 kcal/mol, which is only slightly less than the activation energy for colchicine binding to tubulin. This finding argues against conformational flexibility of the C ring as the structural feature of colchicine responsible for the slow kinetics of colchicinoid-tubulin binding reactions. Tubulin binding promote a dramatic enhancement of allocolchicine fluorescence. Unlike colchicine, the emission energy and intensity of the tubulin-bound allocolchicine fluorescence can be mimicked by solvent, and a general hydrophobic environment for the ligand binding site is indicated. The excitation spectrum of the protein-bound species, however, is shown to possess two bands which center at higher and lower energy than the energy maximum of the spectrum of the ligand in apolar solvents, indicating that properties of the colchicine binding site in addition to a low dielectric constant contribute to the fluorescence of the bound species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Double-mixing kinetic studies of the reactions of methyl isocyanide and CO with diliganded intermediates of hemoglobin: alpha 2CO beta 2 and alpha 2 beta 2CO

Kinetics of the reactions of CO and methyl isocyanide with two diliganded intermediates of hemoglobin, alpha 2CO beta 2 and alpha 2 beta 2CO, have been studied by double-mixing and microperoxidase methods. The valency hybrids were prepared by high-pressure liquid chromatography. The reaction time courses of ligand combination and dissociation with both of the ligands were biphasic, and in CO combination reaction the zero-time amplitudes of the two phases were independent of the protein concentration. In the presence of 2 M urea the reaction time course was clearly dependent on protein concentration, as the zero-time amplitude of the fast phase increased at lower protein concentrations. These two observations indicate that little dissociation of tetramers into dimers occurs in the absence of urea. Consistent with this, the kinetic data for the reactions of CO best fit a reaction model consisting of two tetrameric species not in rapid equilibrium with each other. Various considerations, however, suggest that the reaction model is more appropriately described as 2D in equilibrium R in equilibrium T. The reaction of triliganded species (Hb4(CO)2Me1) with methyl isocyanide was monophasic, and the reaction model suggested a fast T in equilibrium R structural change after the binding of the third ligand. Although the precise structural nature of the two species remains undefined, it is concluded that the biphasicity in the reactions of the two hybrids is characteristic of the diliganded species only and is independent of the nature of the ligand.     

Biophysical and structural analysis of a novel heme B iron ligation in the flavocytochrome cellobiose dehydrogenase

The fungal extracellular flavocytochrome cellobiose dehydrogenase (CDH) participates in lignocellulose degradation. The enzyme has a cytochrome domain connected to a flavin-binding domain by a peptide linker. The cytochrome domain contains a 6-coordinate low spin b-type heme with unusual iron ligands and coordination geometry. Wild type CDH is only the second example of a b-type heme with Met-His ligation, and it is the first example of a Met-His ligation of heme b where the ligands are arranged in a nearly perpendicular orientation. To investigate the ligation further, Met65 was replaced with a histidine to create a bis-histidyl ligated iron typical of b-type cytochromes. The variant is expressed as a stable 90-kDa protein that retains the flavin domain catalytic reactivity. However, the ability of the mutant to reduce external one-electron acceptors such as cytochrome c is impaired. Electrochemical measurements demonstrate a decrease in the redox midpoint potential of the heme by 210 mV. In contrast to the wild type enzyme, the ferric state of the protoheme displays a mixed low spin/high spin state at room temperature and low spin character at 90 K, as determined by resonance Raman spectroscopy. The wild type cytochrome does not bind CO, but the ferrous state of the variant forms a CO complex, although the association rate is very low. The crystal structure of the M65H cytochrome domain has been determined at 1.9 A resolution. The variant structure confirms a bis-histidyl ligation but reveals unusual features. As for the wild type enzyme, the ligands have a nearly perpendicular arrangement. Furthermore, the iron is bound by imidazole N delta 1 and N epsilon 2 nitrogen atoms, rather than the typical N epsilon 2/N epsilon 2 coordination encountered in bis-histidyl ligated heme proteins. To our knowledge, this is the first example of a bis-histidyl N delta 1/N epsilon 2-coordinated protoporphyrin IX iron.     

Structural studies of constitutive nitric oxide synthases with diatomic ligands bound

Crystal structures are reported for the endothelial nitric oxide synthase (eNOS)–arginine–CO ternary complex as well as the neuronal nitric oxide synthase (nNOS) heme domain complexed with l-arginine and diatomic ligands, CO or NO, in the presence of the native cofactor, tetrahydrobiopterin, or its oxidized analogs, dihydrobiopterin and 4-aminobiopterin. The nature of the biopterin has no influence on the diatomic ligand binding. The binding geometries of diatomic ligands to nitric oxide synthase (NOS) follow the {MXY} ⁿ formalism developed from the inorganic diatomic–metal complexes. The structures reveal some subtle structural differences between eNOS and nNOS when CO is bound to the heme which correlate well with the differences in CO stretching frequencies observed by resonance Raman techniques. The detailed hydrogen-bonding geometries depicted in the active site of nNOS structures indicate that it is the ordered active-site water molecule rather than the substrate itself that would most likely serve as a direct proton donor to the diatomic ligands (CO, NO, as well as O₂) bound to the heme. This has important implications for the oxygen activation mechanism critical to NOS catalysis.     

Small ligands that neither bind to nor alter the structure of d(GA·TC)n sequences in DNA

Three minor-groove binding ligands have been used to study the characteristics of two d(GA·CT)_n DNAs embedded in longer DNA fragments. The binding of mithramycin, netropsin or Thia-Net to these sequences has been studied using DNAse I footprinting. None of these ligands appeared to bind to d(GA·CT)₅ nor to d(GA·CT)₂₂ extensively, although with mithramycin some protected bonds were detected at the very edge of these sequences. In general, these small ligands did not enhance the DNAse I cleavage patterns at the alternating d(GA·CT)_n flanking sequences located near DNA regions where the drug was bound. The d(GA·CT)_n sequences could act as a rigid block in which it is not easy to propagate structural changes, whereas other sequences flanking the binding sites showed cleavage enhancements.     

The A-domain of beta 2 integrin CR3 (CD11b/CD18) is a receptor for the hookworm-derived neutrophil adhesion inhibitor NIF

The A-domain is a approximately 200-amino acid peptide present within structurally diverse proadhesive proteins including seven integrins. A recombinant form of the A-domain of beta 2 integrins CR3 and LFA-1 has been recently shown to bind divalent cations and to contain binding sites for protein ligands that play essential roles in leukocyte trafficking to inflammatory sites, phagocytosis and target cell killing. In this report we demonstrate that the neutrophil adhesion inhibitor, NIF produced by the hookworm Ancyclostoma caninium is a selective CD11b A-domain binding protein. NIF bound directly, specifically and with high affinity (Kd of approximately 1 nM) to recombinant CD11b A-domain (r11bA). The binding reaction was characterized by rapid association and very slow dissociation, and was blocked by an anti-r11bA monoclonal antibody. No binding was observed to rCD11aA. The NIF-r11bA interaction required divalent cations, and was absent when the mutant r11bA D140GS/AGA (that lacks divalent cation binding capacity) was used. The NIF binding site in r11bA was mapped to four short peptides, one of which being an iC3b binding site. The interaction of NIF with CR3 in intact cells followed similar binding kinetics to those with r11bA, and occurred with similar affinity in resting and activated human neutrophils, suggesting that the NIF epitope is activation independent. Binding of NIF to CR3 blocked its ability to bind to its ligands iC3b, fibrinogen, and CD54, and inhibited the ability of human neutrophils to ingest serum opsonized particles. NIF thus represents the first example of a disintegrin that targets the integrin A-domain, and is likely to be used by the hookworm to evade the host's inflammatory response. The unique structure of NIF, which lacks a disintegrin motif, emphasizes basic structural differences in antagonists targeting A+ and A- integrins, that should be valuable in drug design efforts aimed at generating novel therapeutics. Identification of the region in NIF mediating A-domain binding should also be useful in this regard, and may, as in the case of disintegrins, unravel a new structural motif with cellular counterparts mediating important physiologic functions.     

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.