Structural characterization of the tunnels of Mycobacterium tuberculosis truncated hemoglobin N from molecular dynamics simulations

The structure of oxygenated trHbN from Mycobacterium tuberculosis shows an extended heme distal hydrogen‐bond network that includes Tyr33(B10), Gln58(E11), and the bound O₂. In addition, trHbN structure shows a network of hydrophobic cavities organized in two orthogonal branches. In the present work, the structure and the dynamics of oxygenated and deoxygenated trHbN in explicit water was investigated from 100 ns molecular dynamics (MD) simulations. Results show that, depending on the presence or the absence of a coordinated O₂, the Tyr33(B10) and Gln58(E11) side chains adopt two different configurations in concert with hydrogen bond network rearrangement. In addition, our data indicate that Tyr33(B10) and Gln58(E11) control the dynamics of Phe62(E15). In deoxy‐trHbN, Phe62(E15) is restricted to one conformation. Upon O₂ binding, the conformation of Gln58(E11) changes and residue Phe62(E15) fluctuates between two conformations. We also conducted a systematic study of trHbN tunnels by analyzing thousands of MD snapshots with CAVER. The results show that tunnel formation is the result of the dynamic reshaping of short‐lived hydrophobic cavities. The analyses indicate that the presence of these cavities is likely linked to the rigid structure of trHbN and also reveal two tunnels, EH and BE, that link the protein surface to the buried distal heme pocket and not present in the crystallographic structure. The cavities are sufficiently large to accomodate and store ligands. Tunnel dynamics in trHbN was found to be controlled by the side‐chain conformation of the Tyr33(B10), Gln58(E11), and Phe62(E15) residues. Importantly, in contrast to recently published works, our extensive systematic studies show that the presence or absence of a coordinated dioxygen does not control the opening of the long tunnel but rather the opening of the EH tunnel. In addition, the data lead to new and distinctly different conclusion on the impact of the Phe62(E15) residue on trHbN tunnels. We propose that the EH and the long tunnels are used for apolar ligands storage. The trajectories bring important new structural insights related to trHbN function and to ligand diffusion in proteins. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Mycobacterium tuberculosis hemoglobin N displays a protein tunnel suited for O2 diffusion to the heme

Macrophage-generated oxygen- and nitrogen-reactive species control the development of Mycobacterium tuberculosis infection in the host. Mycobacterium tuberculosis 'truncated hemoglobin' N (trHbN) has been related to nitric oxide (NO) detoxification, in response to macrophage nitrosative stress, during the bacterium latent infection stage. The three-dimensional structure of oxygenated trHbN, solved at 1.9 A resolution, displays the two-over-two alpha-helical sandwich fold recently characterized in two homologous truncated hemoglobins, featuring an extra N-terminal alpha-helix and homodimeric assembly. In the absence of a polar distal E7 residue, the O2 heme ligand is stabilized by two hydrogen bonds to TyrB10(33). Strikingly, ligand diffusion to the heme in trHbN may occur via an apolar tunnel/cavity system extending for approximately 28 A through the protein matrix, connecting the heme distal cavity to two distinct protein surface sites. This unique structural feature appears to be conserved in several homologous truncated hemoglobins. It is proposed that in trHbN, heme Fe/O2 stereochemistry and the protein matrix tunnel may promote O2/NO chemistry in vivo, as a M.tuberculosis defense mechanism against macrophage nitrosative stress.     

The heme pocket geometry of Lucina pectinata hemoglobin II restricts nitric oxide and peroxide entry: model of ligand control for the design of a stable oxygen carrier

Blood pressure elevation has been attributed in large part to the consumption of nitric oxide (NO) by extracellular hemoglobin (Hb) therapeutics following infusion in humans. We studied NO and hydrogen peroxide (H2O2) oxidative reaction kinetics of monomeric Hbs isolated from the clam Lucina pectinata to probe the effects of their distinctive heme pocket chemistries on ligand controls and heme oxidative stability. HbI (Phe43(CD1), Gln64(E7), Phe29(B10), and Phe68(E11)) reacted with high avidity with NO (k'(ox,NO) = 91 microM-1 s-1), whereas HbII (Phe44(CD1), Gln65(E7), Tyr30(B10), and Phe69(E11)) reacted at a much slower rate (k'(ox,NO)= 2.8 microM-1 s-1). However, replacing B10 (Phe) by Tyr in recombinant HbI (HbI PheB10Tyr) produced only a 2-fold reduction in the NO-induced oxidation rate (k'(ox,NO)= 49.9 microM-1 s-1). Among the clam Hbs, HbII exhibited the fastest NO dissociation and the slowest NO association with ferrous iron. Autoxidation, H2O2-mediated ferryl iron (FeIV) formation, and the subsequent heme degradation kinetics were much slower in HbII and HbI PheB10Tyr when compared to those of HbI. The Tyr(B10) residue appears to afford a greater heme oxidative stability advantage toward H2O2, whereas the close proximity of this residue together with Gln(E7) to the heme iron contributes largely to the distal control of NO binding. Engineering of second-generation Hb-based oxygen therapeutics that are resistant to NO/H2O2-driven oxidation may ultimately require further optimization of the heme pocket architecture to limit heme exposure to solvent.     

Truncated hemoglobins and nitric oxide action

Truncated hemoglobins (trHbs) build a separate subfamily within the hemoglobin superfamily; they are scarcely related by sequence similarity to (non-)vertebrate hemoglobins, displaying amino acid sequences in the 115-130 residue range. The trHb tertiary structure is based on a 2-on-2 alpha-helical sandwich, which hosts a unique hydrophobic cavity/tunnel system, traversing the protein matrix, from the molecular surface to the heme distal site. Such a protein matrix system may provide a path for diffusion of ligands to the heme. In Mycobacterium tuberculosis trHbN the heme-bound oxygen molecule is part of an extended hydrogen bond network including the heme distal residues TyrB10 and GlnE11. In vitro experiments have shown that M. tuberculosis trHbN supports efficiently nitric oxide dioxygenation, yielding nitrate. Such a reaction would provide a defense barrier against the nitrosative stress raised by host macrophages during lung infection. It is proposed that the whole protein architecture, the heme distal site hydrogen bonded network, and the unique protein matrix tunnel, are optimally designed to support the pseudo-catalytic role of trHbN in converting the reactive NO species into the harmless NO3-.     

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

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

Ligand interactions in the distal heme pocket of Mycobacterium tuberculosis truncated hemoglobin N: roles of TyrB10 and GlnE11 residues

The crystallographic structure of oxygenated trHbN from Mycobacterium tuberculosis showed an extended heme distal site hydrogen-bonding network that includes Y(B10), Q(E11), and the bound O(2) (Milani, M., et al. (2001) EMBO J. 20, 3902-3909). In the present work, we analyze the effects that substitutions at the B10 and E11 positions exert on the heme and its coordinated ligands, using steady-state resonance Raman spectroscopy, absorption spectroscopy and X-ray crystallography. Our results show that (1) residues Y(B10) and Q(E11) control the binding and the ionization state of the heme-bound water molecules in ferric trHbN and are important in keeping the sixth coordination position vacant in deoxy trHbN; (2) residue Q(E11) plays a role in maintaining the integrity of the proximal Fe-His bond in deoxy trHbN; (3) in wild-type oxy-trHbN, the size and hydrogen-bonding capability of residue E11 is important to sustain proper interaction between Y(B10) and the heme-bound O(2); (4) CO-trHbN is in a conformational equilibrium, where either the Y(B10) or the Q(E11) residue interacts with the heme-bound CO; and (5) Y(B10) and Q(E11) residues control the conformation (and likely the dynamics) of the protein matrix tunnel gating residue F(E15). These findings suggest that the functional processes of ligand binding and diffusion are controlled in trHbN through the dynamic interaction of residues Y(B10), Q(E11), F(E15), and the heme ligand.     

NO binding induced conformational changes in a truncated hemoglobin from Mycobacterium tuberculosis

The resonance Raman spectra of the NO-bound ferric derivatives of wild-type HbN and the B10 Tyr --> Phe mutant of HbN, a hemoglobin from Mycobacterium tuberculosis, were examined with both Soret and UV excitation. The Fe-N-O stretching and bending modes of the NO derivative of the wild-type protein were tentatively assigned at 591 and 579 cm(-1), respectively. Upon B10 mutation, the Fe-NO stretching mode was slightly enhanced and the bending mode diminished in amplitude. In addition, the N-O stretching mode shifted from 1914 to 1908 cm(-1). These data suggest that the B10 Tyr forms an H-bond(s) with the heme-bound NO and causes it to bend in the wild-type protein. To further investigate the interaction between the B10 Tyr and the heme-bound NO, we examined the UV Raman spectrum of the B10 Tyr by subtracting the B10 mutant spectrum from the wild-type spectrum. It was found that, upon NO binding to the ferric protein, the Y(8a) mode of the B10 Tyr shifted from 1616 to 1622 cm(-1), confirming a direct interaction between the B10 Tyr and the heme-bound NO. Furthermore, the Y(8a) mode of the other two Tyr residues at positions 16 and 72 that are remote from the heme was also affected by NO binding, suggesting that NO binding to the distal site of the heme triggers a large-scale conformational change that propagates through the pre-F helix loop to the E and B helices. This large-scale conformational change triggered by NO binding may play an important role in regulating the ligand binding properties and/or the chemical reactivity of HbN.     

The truncated hemoglobin from Mycobacterium leprae

Truncated hemoglobins (trHb's) form a family of low molecular weight O2 binding hemoproteins distributed in eubacteria, protozoa, and plants. TrHb's branch in a distinct clade within the hemoglobin (Hb) superfamily. A unique globin gene has recently been identified from the complete genome sequence of Mycobacterium leprae that is predicted to encode a trHb (M. leprae trHbO). Sequence comparison and modelling considerations indicate that monomeric M. leprae trHbO has structural features typical of trHb's, such as 20-40 fewer residues than conventional globin chains, Gly-based sequence consensus motifs, likely assembling into a 2-on-2 alpha-helical sandwich fold, and hydrophobic residues recognized to build up the protein matrix ligand diffusion tunnel. The ferrous heme iron atom of deoxygenated M. leprae trHbO appears to be hexacoordinated, like in Arabidopsis thaliana trHbO-3 (A. thaliana trHbO-3). Accordingly, the value of the second-order rate constant for M. leprae trHbO carbonylation (7.3 x 10(3) M(-1) s(-1)) is similar to that observed for A. thaliana trHbO-3 (1.4 x 10(4) M(-1) s(-1)) and turns out to be lower than that reported for carbon monoxide binding to pentacoordinated Mycobacterium tuberculosis trHbN (6.7 x 10(6) M(-1) s(-1)). The lower reactivity of M. leprae trHbO as compared to M. tuberculosis trHbN might be related to the higher susceptibility of the leprosy bacillus to toxic nitrogen and oxygen species produced by phagocytic cells.     

Analysis of the kinetic barriers for ligand binding to sperm whale myoglobin using site-directed mutagenesis and laser photolysis techniques

Time courses for NO, O2, CO, methyl and ethyl isocyanide rebinding to native and mutant sperm whale myoglobins were measured at 20 degrees C following 17-ns and 35-ps laser excitation pulses. His64 (E7) was replaced with Gly, Val, Leu, Phe, and Gln, and Val68 (E11) was replaced with Ala, Ile, and Phe. For both NO and O2, the effective picosecond quantum yield of unliganded geminate intermediates was roughly 0.2 and independent of the amino acids at positions 64 and 68. Geminate recombination of NO was very rapid; 90% rebinding occurred within 0.5-1.0 ns for all of the myoglobins examined; and except for the Gly64 and Ile68 mutants, the fitted recombination rate parameters were little influenced by the size and polarity of the amino acid at position 64 and the size of the residue at position 68. The rates of NO recombination and ligand movement away from the iron atom in the Gly64 mutant increased 3-4-fold relative to native myoglobin. For Ile68 myoglobin, the first geminate rate constant for NO rebinding decreased approximately 6-fold, from 2.3 x 10(10) s-1 for native myoglobin to 3.8 x 10(9) s-1 for the mutant. No picosecond rebinding processes were observed for O2, CO, and isocyanide rebinding to native and mutant myoglobins; all of the observed geminate rate constants were less than or equal to 3 x 10(8) s-1. The rebinding time courses for these ligands were analyzed in terms of a two-step consecutive reaction scheme, with an outer kinetic barrier representing ligand movement into and out of the protein and an inner barrier representing binding to the heme iron atom by ligand occupying the distal portion of the heme pocket. Substitution of apolar amino acids for His64 decreased the absolute free energies of the outer and inner kinetic barriers and the well for non-covalently bound O2 and CO by 1 to 1.5 kcal/mol, regardless of size. In contrast, the His64 to Gln mutation caused little change in the barrier heights for all ligands, showing that the polar nature of His64 inhibits both the bimolecular rate of ligand entry into myoglobin and the unimolecular rate of binding to the iron atom from within the protein. Increasing the size of the position 68(E11) residue in the series Ala to Val (native) to Ile caused little change in the rate of O2 migration into myoglobin or the equilibrium constant for noncovalent binding but did decrease the unimolecular rate for iron-O2 bond formation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Coordination structure of the ferric heme iron in engineered distal histidine myoglobin mutants.

Recombinant human myoglobin mutants with the distal His residue (E7, His64) replaced by Leu, Val, or Gln residues were prepared by site-directed mutagenesis and expression in Escherichia coli. Electronic and coordination structures of the ferric heme iron in the recombinant myoglobin proteins were examined by optical absorption, EPR, 1H NMR, magnetic circular dichroism, and x-ray spectroscopy. Mutations, His-->Val and His-->Leu, remove the heme-bound water molecule resulting in a five-coordinate heme iron at neutral pH, while the heme-bound water molecule appears to be retained in the engineered myoglobin with His-->Gln substitution as in the wild-type protein. The distal Val and distal Leu ferric myoglobin mutants at neutral pH exhibited EPR spectra with g perpendicular values smaller than 6, which could be interpreted as an admixture of intermediate (S = 3/2) and high (S = 5/2) spin states. At alkaline pH, the distal Gln mutant is in the same so-called "hydroxy low spin" form as the wild-type protein, while the distal Leu and distal Val mutants are in high spin states. The ligand binding properties of these recombinant myoglobin proteins were studied by measurements of azide equilibrium and cyanide binding. The distal Leu and distal Val mutants exhibited diminished azide affinity and extremely slow cyanide binding, while the distal Gln mutant showed azide affinity and cyanide association rate constants similar to those of the wild-type protein.     

Ligand-induced dynamical regulation of NO conversion in Mycobacterium tuberculosis truncated hemoglobin-N

Mycobacterium tuberculosis, the causative agent of human tuberculosis, is forced into latency by nitric oxide produced by macrophages during infection. In response to nitrosative stress M. tuberculosis has evolved a defense mechanism that relies on the oxygenated form of "truncated hemoglobin" N (trHbN), formally acting as NO-dioxygenase, yielding the harmless nitrate ion. X-ray crystal structures have shown that trHbN hosts a two-branched protein matrix tunnel system, proposed to control diatomic ligand migration to the heme, as the rate-limiting step in NO conversion to nitrate. Extended molecular dynamics simulations (0.1 micros), employed here to characterize the factors controlling diatomic ligand diffusion through the apolar tunnel system, suggest that O2 migration in deoxy-trHbN is restricted to a short branch of the tunnel, and that O2 binding to the heme drives conformational and dynamical fluctuations promoting NO migration through the long tunnel branch. The simulation results suggest that trHbN has evolved a dual-path mechanism for migration of O2 and NO to the heme, to achieve the most efficient NO detoxification.     

Cyanide binding to truncated hemoglobins: a crystallographic and kinetic study

Cyanide is one of the few diatomic ligands able to interact with the ferric and ferrous heme-Fe atom. Here, the X-ray crystal structure of the cyanide derivative of ferric Mycobacterium tuberculosis truncated hemoglobin-N (M. tuberculosis trHbN) has been determined at 2.0 A (R-general = 17.8% and R-free = 23.5%), and analyzed in parallel with those of M. tuberculosis truncated hemoglobin-O (M. tuberculosis trHbO), Chlamydomonas eugametos truncated hemoglobin (C. eugametos trHb), and sperm whale myoglobin, generally taken as a molecular model. Cyanide binding to M. tuberculosis trHbN is stabilized directly by residue TyrB10(33), which may assist the deprotonation of the incoming ligand and the protonation of the outcoming cyanide. In M. tuberculosis trHbO and in C. eugametos trHb the ligand is stabilized by the distal pocket residues TyrCD1(36) and TrpG8(88), and by the TyrB10(20) - GlnE7(41) - GlnE11(45) triad, respectively. Moreover, kinetics for cyanide binding to ferric M. tuberculosis trHbN and trHbO and C. eugametos trHb, for ligand dissociation from the ferrous trHbs, and for the reduction of the heme-Fe(III)-cyanide complex have been determined, at pH 7.0 and 20.0 degrees C. Despite the different heme distal site structures and ligand interactions, values of the rate constant for cyanide binding to ferric (non)vertebrate heme proteins are similar, being influenced mainly by the presence in the heme pocket of proton acceptor group(s), whose function is to assist the deprotonation of the incoming ligand (i.e., HCN). On the other hand, values of the rate constant for the reduction of the heme-Fe(III)-cyanide (non)vertebrate globins span over several orders of magnitude, reflecting the different ability of the heme proteins considered to give productive complex(es) with dithionite or its reducing species SO(2)(-). Furthermore, values of the rate constant for ligand dissociation from heme-Fe(II)-cyanide (non)vertebrate heme proteins are very different, reflecting the different nature and geometry of the heme distal residue(s) hydrogen-bonded to the heme-bound cyanide.     

Water penetration and binding to ferric myoglobin

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

Reaction of carbon monoxide with the reduced active site of bacterial nitric oxide reductase.

Bacterial nitric oxide reductase (NOR), a member of the superfamily of heme-copper oxidases, catalyzes the two-electron reduction of nitric oxide to nitrous oxide. The key feature that distinguishes NOR from the typical heme-copper oxidases is the elemental composition of the dinuclear center, which contains non-heme iron (FeB) rather than copper (CuB). UV-vis electronic absorption and room-temperature magnetic circular dichroism (RT-MCD) spectroscopies showed that CO binds to Fe(II) heme b3 to yield a low-spin six-coordinate species. Photolysis of the Fe(II)-CO bond is followed by CO recombination (k(on) = 1.7 x 10(8) M(-1) x s(-1)) that is approximately 3 orders of magnitude faster than CO recombination to the active site of typical heme-copper oxidases (k(on) = 7 x 10(4) M(-1)x s(-1)). This rapid rate of CO recombination suggests an unimpeded pathway to the active site that may account for the enzyme's high affinity for substrate, essential for maintaining denitrification at low concentrations of NO. In contrast, the initial binding of CO to reduced heme b3 measured by stopped-flow spectroscopy is much slower (k(on) = 1.2 x 10(5) M(-1) x s(-1)). This suggests that an existing heme distal ligand (water/OH-) may be displaced to elicit the spin-state change observed in the RT-MCD spectrum.     

Six- to five-coordinate heme-nitrosyl conversion in cytochrome c' and its relevance to guanylate cyclase

The 5-coordinate ferrous heme of Alcaligenes xylosoxidans cytochrome c' reacts with NO to form a 6-coordinate nitrosyl intermediate (lambdaSoret at 415 nm) which subsequently converts to a 5-coordinate nitrosyl end product (lambdaSoret at 395 nm) in a rate-determining step. Stopped-flow measurements at pH 8.9, 25 degrees C, yield a rate constant for the formation of the 6-coordinate nitrosyl adduct, k(on) = (4.4 +/- 0.5) x 10(4) M(-1) x s(-1), which is 3-4 orders of magnitude lower than the values for other pentacoordinate ferrous hemes and is consistent with NO binding within the sterically crowded distal heme pocket. Resonance Raman measurements of the freeze-trapped 6-coordinate nitrosyl intermediate reveal an unusually high Fe-NO stretching frequency of 579 cm(-1), suggesting a distorted Fe-N-O coordination geometry. The rate of 6- to 5-coordinate heme nitrosyl conversion is also dependent upon NO concentration, with a rate constant, k(6-5) = (8.1 +/- 0.7) x 10(3) M(-1) x s(-1), implying that an additional molecule of NO is required to form the 5c-NO adduct. Since crystallographic studies have shown that the 5-coordinate nitrosyl complex of cytochrome c' binds NO to the proximal (rather than distal) face of the heme, the NO dependence of the 6- to 5-coordinate NO conversion supports a mechanism in which the weakened His ligand, as well as the distally bound NO, is displaced by a second NO molecule which attacks and is retained in the proximal coordination position. The fact that a dependent 6- to 5-coordinate nitrosyl conversion has been previously reported for soluble guanylate cyclase suggests that the mechanism of Fe-His bond cleavage may be similar to that of cytochrome c' and strengthens the recent proposal that both proteins exhibit proximal NO binding in their 5-coordinate nitrosyl adducts.     

Theoretical Investigations of Nitric Oxide Channeling in Mycobacterium tuberculosis Truncated Hemoglobin N

Mycobacterium tuberculosis group I truncated hemoglobin trHbN catalyzes the oxidation of nitric oxide (•NO) to nitrate with a second-order rate constant k ≈ 745 μM⁻¹ s⁻¹ at 23°C (nitric oxide dioxygenase reaction). It was proposed that this high efficiency is associated with the presence of hydrophobic tunnels inside trHbN structure that allow substrate diffusion to the distal heme pocket. In this work, we investigated the mechanisms of •NO diffusion within trHbN tunnels in the context of the nitric oxide dioxygenase reaction using two independent approaches. Molecular dynamics simulations of trHbN were performed in the presence of explicit •NO molecules. Successful •NO diffusion from the bulk solvent to the distal heme pocket was observed in all simulations performed. The simulations revealed that •NO interacts with trHbN at specific surface sites, composed of hydrophobic residues located at tunnel entrances. The entry and the internal diffusion of •NO inside trHbN were performed using the Long, Short, and EH tunnels reported earlier. The Short tunnel was preferentially used by •NO to reach the distal heme pocket. This preference is ascribed to its hydrophobic funnel-shape entrance, covering a large area extending far from the tunnel entrance. This funnel-shape entrance triggers the frequent formation of solvent-excluded cavities capable of hosting up to three •NO molecules, thereby accelerating •NO capture and entry. The importance of hydrophobicity of entrances for •NO capture is highlighted by a comparison with a polar mutant for which residues at entrances were mutated with polar residues. A complete map of •NO diffusion pathways inside trHbN matrix was calculated, and •NO molecules were found to diffuse from Xe cavity to Xe cavity. This scheme was in perfect agreement with the three-dimensional free-energy distribution calculated using implicit ligand sampling. The trajectories showed that •NO significantly alters the dynamics of the key amino acids of Phe⁶²(E15), a residue proposed to act as a gate controlling ligand traffic inside the Long tunnel, and also of Ile¹¹⁹(H11), at the entrance of the Short tunnel. It is noteworthy that •NO diffusion inside trHbN tunnels is much faster than that reported previously for myoglobin. The results presented in this work shed light on the diffusion mechanism of apolar gaseous substrates inside protein matrix.     

X-ray crystallographic study of cyanide binding provides insights into the structure-function relationship for cytochrome cd1 nitrite reductase from Paracoccus pantotrophus

We present a 1.59-A resolution crystal structure of reduced Paracoccus pantotrophus cytochrome cd(1) with cyanide bound to the d(1) heme and His/Met coordination of the c heme. Fe-C-N bond angles are 146 degrees for the A subunit and 164 degrees for the B subunit of the dimer. The nitrogen atom of bound cyanide is within hydrogen bonding distance of His(345) and His(388) and either a water molecule in subunit A or Tyr(25) in subunit B. The ferrous heme-cyanide complex is unusually stable (K(d) approximately 10(-6) m); we propose that this reflects both the design of the specialized d(1) heme ring and a general feature of anion reductases with active site heme. Oxidation of crystals of reduced, cyanide-bound, cytochrome cd(1) results in loss of cyanide and return to the native structure with Tyr(25) as a ligand to the d(1) heme iron and switching to His/His coordination at the c-type heme. No reason for unusually weak binding of cyanide to the ferric state can be identified; rather it is argued that the protein is designed such that a chelate-based effect drives displacement by tyrosine of cyanide or a weaker ligand, like reaction product nitric oxide, from the ferric d(1) heme.     

Structure and ligand selection of hemoglobin II from Lucina pectinata

Lucina pectinata ctenidia harbor three heme proteins: sulfide-reactive hemoglobin I (HbI(Lp)) and the oxygen transporting hemoglobins II and III (HbII(Lp) and HbIII(Lp)) that remain unaffected by the presence of H(2)S. The mechanisms used by these three proteins for their function, including ligand control, remain unknown. The crystal structure of oxygen-bound HbII(Lp) shows a dimeric oxyHbII(Lp) where oxygen is tightly anchored to the heme through hydrogen bonds with Tyr(30)(B10) and Gln(65)(E7). The heme group is buried farther within HbII(Lp) than in HbI(Lp). The proximal His(97)(F8) is hydrogen bonded to a water molecule, which interacts electrostatically with a propionate group, resulting in a Fe-His vibration at 211 cm(-1). The combined effects of the HbII(Lp) small heme pocket, the hydrogen bonding network, the His(97) trans-effect, and the orientation of the oxygen molecule confer stability to the oxy-HbII(Lp) complex. Oxidation of HbI(Lp) Phe(B10) --> Tyr and HbII(Lp) only occurs when the pH is decreased from pH 7.5 to 5.0. Structural and resonance Raman spectroscopy studies suggest that HbII(Lp) oxygen binding and transport to the host bacteria may be regulated by the dynamic displacements of the Gln(65)(E7) and Tyr(30)(B10) pair toward the heme to protect it from changes in the heme oxidation state from Fe(II) to Fe(III).     

The 2.6-A crystal structure of Pseudomonas putida cytochrome P-450

The crystal structure of Pseudomonas putida cytochrome P-450cam in the ferric, camphor bound form has been determined and partially refined to R = 0.23 at 2.6 A. The single 414 amino acid polypeptide chain (Mr = 45,000) approximates a triangular prism with a maximum dimension of approximately 60 A and a minimum of approximately 30 A. Twelve helical segments (A through L) account for approximately 40% of the structure while antiparallel beta pairs account for only approximately 10%. The unexposed iron protoporphyrin IX is sandwiched between two parallel helices designated the proximal and distal helices. The heme iron atom is pentacoordinate with the axial sulfur ligand provided by Cys 357 which extends from the N-terminal end of the proximal (L) helix. A substrate molecule, 2-bornanone (camphor), is buried in an internal pocket just above the heme distal surface adjacent to the oxygen binding site. The substrate molecule is held in place by a hydrogen bond between the side chain hydroxyl group of Tyr 96 and the camphor carbonyl oxygen atom in addition to complementary hydrophobic contacts between the camphor molecule and neighboring aliphatic and aromatic residues. The camphor is oriented such that the exo-surface of C5 would contact an iron bound, "activated" oxygen atom for stereoselective hydroxylation.     

Thermodynamics of heme binding to the HasA(SM) hemophore: effect of mutations at three key residues for heme uptake

HasA(SM) secreted by the Gram-negative bacterium Serratia marcescens belongs to the hemophore family. Its role is to take up heme from host heme carriers and to shuttle it to specific receptors. Heme is linked to the HasA(SM) protein by an unusual axial ligand pair: His32 and Tyr75. The nucleophilic nature of the tyrosine is enhanced by the hydrogen bonding of the tyrosinate to a neighboring histidine in the binding site: His83. We used isothermal titration microcalorimetry to examine the thermodynamics of heme binding to HasA(SM) and showed that binding is strongly exothermic and enthalpy driven: DeltaH = -105.4 kJ x mol(-1) and TDeltaS = -44.3 kJ x mol(-1). We used displacement experiments to determine the affinity constant of HasA(SM) for heme (K(a) = 5.3 x 10(10) M(-1)). This is the first time that this has been reported for a hemophore. We also analyzed the thermodynamics of the interaction between heme and a panel of single, double, and triple mutants of the two axial ligands His32 and Tyr75 and of His83 to assess the implication of each of these three residues in heme binding. We demonstrated that, in contrast to His32, His83 is essential for the binding of heme to HasA(SM), even though it is not directly coordinated to iron, and that the Tyr75/His83 pair plays a key role in the interaction.