Heme Binding Pocket Research Articles

Hb Dothan [β25/26 (B7/B8)/(-GTG/-GLY)/Gly+Glu→Glu]; A Novel Mechanism Leading to a M-Hemoglobin.

Hereditary methemoglobinemia is a relatively rare disorder usually manifesting with cyanosis at birth. The more common form results from the deficiency of the enzyme, NADH-Cytochrome b5 reductase (methemoglobin reductase, diaphorase) and displays an autosomal recessive inheritance pattern. Less common are the so-called M-hemoglobins with an autosomal dominant pattern, which result from amino acid substitutions in the heme binding pocket of α, β, or less commonly γ-globin chains. The majority of the M-hemoglobin (Hb) variants occur from substitutions in the E or F-helices, which constitute the heme binding pocket, most commonly from amino acid substitutions involving the conserved proximal (F8) or distal (E11) histidine residues. Here we report a new Hb variant due to a three nucleotide deletion (-GTG between codons 25 and 26 of the β globin gene causing a single amino acid (-Gly) deletion in the B helix (B7/B8) of the β-globin chain that leads to methemoglobinemia with a novel mechanism. The propositus is a 9 month old Caucasian boy from Dothan AL who was found to have a low O2 saturation prior to an ENT procedure. He was referred to cardiology at Children's Health System, Birmingham, AL to rule out a congenital heart disease. A low O2 saturation (85–86%) was confirmed. Cardiac catheterization excluded the structural abnormality of the heart. Cooximetry showed a normal PaO2 but confirmed a low O2 saturation. Methemoglobin level was 20%, while methemoglobin reductase activity was in the low–normal range but when repeated was found to be normal. His growth and development have been normal. On alkaline electrophoresis an abnormal hemoglobin band was observed. The patient's blood was sent to the Hemoglobinopathy Laboratory of the Sickle Cell Center at MCG, Augusta, GA for definite identification of the variant. CBC revealed a RBC of 4.3 M/mm3, HGB 13.6 g/dL, HCT 40.8 %, MCV 95.2 fl, MCH 31.8 pg MCHC 33.4 g/dL, Retics 4.4 %. Isoelectrofocusing (IEF) on agarose showed the presence of an abnormal Hb with approximately the same isoelectric point (pI) as Hb F. Quantitation of Hb components by Cation Exchange HPLC revealed 62.7% Hb A, 27.9% Hb X, 3.0% Hb A2, and 6.4% Hb F. By globin chain analyses with reversed phase HPLC, βχ was detected as 37.6% of the total beta chains. Isopropanol stability test gave strongly positive results. P50 was found to be 24.8 mm Hg in the patient and 26.4 in the control (slightly increased oxygen affinity). Peptide analysis was done using mass spectrometry (Alphalyse, Palo Alto, CA) where tryptic digests of purified Hb X (95.0% enriched) and normal control (97.0% Hb A) were analyzed and compared. Peptide 19–30 of helix-B fragment revealed 1314 Da mass in control, whereas peptide 19–29 (with –Gly) of helix-B fragment of Hb X gave 1257 Da mass, confirming the deletion of a Gly residue. The corresponding deletion of three nucleotides (-GTG) in the genomic DNA (codons 25–26: GTGGAG→GAG ) was demonstrated by polymerase chain reaction (PCR) amplification and direct sequencing of β-globin gene and confirmed by cDNA sequencing of β-globin mRNA. No abnormality was detected in the sequences of δ, Gγ, Aγ, α1 and α2 globin genes. The three nucleotide deletion between codons 25 and 26 (-GTG) of the β-globin gene causes a one amino acid (-Gly) deletion in the B helix (B7/B8) of the β-globin chain, however does not alter the amino acid composition of β-globin chain after the deletion point but results in a shorter (145 AA, instead of the normal 146) mutant β-globin chain. As a result close spatial contact of amino acids in tertiary structure of hemoglobin is altered completely. Most importantly, distal histidine at residue 63 of E7 helix now becomes Gly leading to methemoglobin formation. A similar variant was previously reported in a Japanese baby, Hb Higashitochigi (Fujisawa et al, Hemoglobin , 17:467, 1993) where a three nucleotide deletion in codons 24/25 also resulted in the loss of a single Gly residue with a similar outcome. These two cases differ from the known M-hemoglobins all of which result from single amino acid substitution in the E or F-helices thus altering the heme pocket. Hb Dothan and Hb Higashitochigi represent a novel mechanism for M-hemoglobin generation where an in frame deletion alters the tertiary structure of the globin chain with alterations in the structure of E-helix and loss of the distal histidine residue.

Conformational Dynamics Associated with Photodissociation of CO from Dehaloperoxidase Studied Using Photoacoustic Calorimetry

Herein, we present photoacoustic calorimetry and transient absorption studies of the dynamics and energetics associated with dissociation of a ligand from Fe(2+) dehaloperoxidase (DHP) from Amphitrite ornata. Our data show that CO photodissociation is associated with an endothermic (DeltaH = 8 +/- 3 kcal mol(-1)) volume expansion (DeltaV = 9.4 +/- 0.6 mL mol(-1)) that occurs within 50 ns upon photodissociation. No additional thermodynamics were detected on slower time scales (up to 10 micros), suggesting that the dissociated ligand rapidly escapes from the heme-binding pocket into the surrounding solvent. Similar volume and enthalpy changes were observed for CO photodissociation in the presence of the substrate, 2,4-dichlorophenol or 4-bromophenol, indicating that either the substrate does not bind in the protein distal cavity at ambient temperature or its presence does not impact the thermodynamic profile associated with ligand dissociation. We attribute a fast ligand exchange between the protein active site and the surrounding solvent to the high flexibility of the distal histidine residue, His55, that provides a direct pathway between the heme-binding pocket and the protein exterior. The dynamics and energetics of conformational changes observed for dissociation of a ligand from DHP differ significantly from those measured previously for photodissociation of CO from the structural homologue myoglobin, suggesting that structural dynamics in DHP are fine-tuned to enhance the peroxidase function of this protein.

Comparison of Apo- and Heme-bound Crystal Structures of a Truncated Human Heme Oxygenase-2

Heme oxygenase (HO) catalyzes the first step in the heme degradation pathway. The crystal structures of apo- and heme-bound truncated human HO-2 reveal a primarily alpha-helical architecture similar to that of human HO-1 and other known HOs. Proper orientation of heme in HO-2 is required for the regioselective oxidation of the alpha-mesocarbon. This is accomplished by interactions within the heme binding pocket, which is made up of two helices. The iron coordinating residue, His(45), resides on the proximal helix. The distal helix contains highly conserved glycine residues that allow the helix to flex and interact with the bound heme. Tyr(154), Lys(199), and Arg(203) orient the heme through direct interactions with the heme propionates. The rearrangements of side chains in heme-bound HO-2 compared with apoHO-2 further elucidate HO-2 heme interactions.

Inhibition of Nitric Oxide Synthase (NOS) 1, NOS2, and NOS3 Enzymatic Activities by Vitamin B12 and Related Corrins: Biochemical and Molecular Modeling Studies.

Cobalamins (Cbl) serve as important co-factors for methionine synthase and methylmalonyl-coA mutase. They also can block the activities of NO, as the cobalt in cobalamin binds NO and quenches its bioactivity. NO plays very important roles in normal physiology and in various pathologic processes. NO is synthesized by NO synthases (NOS), enzymes that convert L-arginine to L-citrulline and NO. We postulated that various corrin derivatives (e.g., cobalamins or cobinamides) would inhibit the enzymatic activity of NO synthase (NOS) by directly interacting with NOS. We used purified, recombinant neuronal NOS (NOS1), inducible NOS (NOS2), and endothelial NOS (NOS3) in in vitro assays measuring conversion of 14-C-L-arginine to 14-C-L-citrulline. Results showed that hydroxocobalamin (OH–Cbl), cobinamide (Cbi), and dicyanocobinamide (CN2–Cbi) were potent inhibitors of all three isoforms of NOS, while cyanocobalamin had much less activity (Table). In general, NOS1 was most inhibited, with affinities generally NOS1>NOS3>NOS2. Some corrins were extremely potent, with the ID50 of CN2-Cbi in the nM range. Methylcobalamin and adenosylcobalamin had very little ability to inhibit NOS activity, but light illumination of these two compounds (a process that liberates the methyl and adenosyl groups from cobalamin, generating OH–Cbl) “activated” them so that they had inhibitory activity comparable to native OH–Cbl. We examined the interactions of NOS2 with cobalamins and cobinamides by spectroscopy. Oxygen binding to the NOS heme iron in the ferrous state is an obligatory step in the NO synthase catalytic cycle. Carbon monoxide (CO, which serves as an oxygen mimic) causes a marked change in the NOS spectrum under reducing conditions. CN2–Cbi markedly diminished the CO-induced spectral changes in NOS2, indicating that CN2–Cbi likely blocks binding of CO to the NOS2 oxygenase domain under reduced conditions. This suggests that CN2–Cbi-mediated inhibition of NOS activity is due to blocking of oxygen binding to heme, a step required for the NOS catalytic cycle. Our molecular modeling investigations using manual computer-assisted docking indicate that the corrins can physically access the unusually large heme and substrate binding pocket of NOS. Binding appears to be best in the “base-off” conformation of the lower axial ligand dimethylbenzimidazole, with binding being facilitated by heme ring-corrin ring interactions and possibly by hydrogen bonding to arginine-binding groups in the active site pocket. The total “base-off” structure of CN2–Cbi and the great potential of OH–Cbl to hydrogen-bond in the pocket help explain their low ID50s for inhibition. The slightly larger active site pocket of NOS1 compared to NOS2 and NOS3 likely explains the lower ID50s for NOS1. While certain cobalamins and cobinamides have been previously noted to bind and quench/scavenge the actions of preformed NO, we show here for the first time that cobalamins and cobinamides can potently inhibit the enzymatic function of all NOS isoforms. These agents (or their derivatives) might serve as NOS inhibitors for treatment of various conditions. Also, native tissue corrins might act as important regulators of NOS activity in normal and pathological conditions in vivo.Dose (uM) for 50% inhibition of NOS activity (ID50)OH–CblCbiCN2–CbiCN–CblMe–CblAdo–CblNOS16150.272576498NOS221502162023102148NOS39430.3519

Evidence That Heme Export Via the Feline Leukemia Virus, Subgroup, C Receptor (FLVCR) Is Carrier Dependent and Its Physiological Implications.

FLVCR is a heme export protein that is required for proerythroblast survival (Cell 18:757, 2004), macrophage heme-iron recycling, and systemic iron homeostasis (ASH abstract, 2007). However, the mechanism and regulation of export through this and related major facilitator superfamily (MFS) members (e.g., Glut4, GlpT) is unknown. To gain insights into the structure and function of FLVCR, we studied the export of zinc mesoporphyrin (ZnMP), a fluorescent heme analog that traffics like heme and blocks heme oxygenase (HO) activity. NRK cells engineered to express huFLVCR were loaded with ZnMP for 30 min, then incubated at 37°C × 90 min with no carrier protein, albumin, or hemopexin (HPXN). In the absence of carrier protein, no export was seen. Export was near complete in the presence of 152 μM (1%) albumin, but less extensive with 15.2 μM albumin and absent with 1.52 μM albumin. Interestingly, HPXN appeared 100 fold more efficacious in inducing ZnMP export, as the residual ZnMP fluorescence in the presence of 0.152 μM HPXN was equivalent to residual intensity in the presence of 15.2 μM albumin. Comparable results were obtained in studies of human blood macrophages. Heme trafficking was next quantitated using 55Fe-heme and a similar study design (ZnMP was added to block HO activity). In the presence of 1.52 μM albumin, 6% of the 55Fe-heme load was exported, while 52% was exported in the presence of 1.52 μM HPXN. When the albumin concentration was increased 100 fold to 152 μM, 67% was exported. As Kds are ∼10 nM and <1 pM, for albumin and HPXN, respectively, our data argue that the rate of heme export depends on the avidity of this binding. Carrier proteins coordinate the heme-iron through two histidines and stabilize this interaction with multiple aromatic amino acids lining the heme-binding pocket. Of note, there are three conserved histidine residues in FLVCR, two of which (aa 145 and 198) are on adjacent extracellular loops, and based upon known MFS family member structures (Protein Sci. 13:1832, 2004), are potentially optimally located to coordinate the heme-iron exiting the channel. Adjacent transmembrane regions contain several aromatic residues capable of lining a heme pocket. These histidine residues are conserved in human, cat, and mouse FLVCR, but are not conserved in closely related paralogs (Gene 286:203, 2002), which lack heme export function. We hypothesize they act as a temporary docking site for heme after it exits through the transport channel and prior to being picked up by HPXN in the circulation, and thus as a structural regulator of export efficiency. We anticipate that when heme binds to this site, the channel is held in the open-out position preventing additional heme from being transported. We are currently using site-directed mutagenesis to generate a mutant FLVCR lacking these two histidines and will test its export function. To our knowledge, comparable regulatory mechanisms have not been previously described for other MFS family members or for transport proteins more broadly. Since HPXN is present in a high concentration in human serum (17–25 μM, similar to transferrin, 22–31 μM), it may function physiologically to facilitate the recycling of heme-iron from macrophages after they ingest senescent red cells, in addition to its role as a scavenger protein for protection from internal bleeding or extravascular hemolysis. By extension, modulation of the serum HPXN concentration might ameliorate the anemia of chronic inflammation.

Novel cholesterol biosynthesis inhibitors targeting human lanosterol 14α-demethylase (CYP51)

Novel cholesterol biosynthesis inhibitors, a group of pyridylethanol(phenylethyl)amine derivatives, were synthesized. Sterol profiling assay in the human hepatoma HepG2 cells revealed that compounds target human lanosterol 14α-demethylase (CYP51). Structure–activity relationship study of the binding with the overexpressed human CYP51 indicates that the pyridine binds within the heme binding pocket in an analogy with the azoles.

Heme Coordination by Staphylococcus aureus IsdE

Staphylococcus aureus is a Gram-positive bacterial pathogen and a leading cause of hospital acquired infections. Because the free iron concentration in the human body is too low to support growth, S. aureus must acquire iron from host sources. Heme iron is the most prevalent iron reservoir in the human body and a predominant source of iron for S. aureus. The iron-regulated surface determinant (Isd) system removes heme from host heme proteins and transfers it to IsdE, the cognate substrate-binding lipoprotein of an ATP-binding cassette transporter, for import and subsequent degradation. Herein, we report the crystal structure of the soluble portion of the IsdE lipoprotein in complex with heme. The structure reveals a bi-lobed topology formed by an N- and C-terminal domain bridged by a single alpha-helix. The structure places IsdE as a member of the helical backbone metal receptor superfamily. A six-coordinate heme molecule is bound in the groove established at the domain interface, and the heme iron is coordinated in a novel fashion for heme transporters by Met(78) and His(229). Both heme propionate groups are secured by H-bonds to IsdE main chain and side chain groups. Of these residues, His(229) is essential for IsdE-mediated heme uptake by S. aureus when growth on heme as a sole iron source is measured. Multiple sequence alignments of homologues from several other Gram-positive bacteria, including the human pathogens pyogenes, Bacillus anthracis, and Listeria monocytogenes, suggest that these other systems function equivalently to S. aureus IsdE with respect to heme binding and transport.

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.

Nonequilibrium Dynamics Simulations of Nitric Oxide Release: Comparative Study of Nitrophorin and Myoglobin

Nitrophorin 4 (NP4) is a heme protein that reversibly binds nitric oxide (NO), with release rates modulated by pH change. High-resolution structures of NP4 revealed that pH changes and NO binding induce a large conformational rearrangement in two loops that serve to protect the heme-bound NO molecule from solvent. We used extended (110 ns) molecular dynamics simulations of NP4 at pH 5 and pH 7, modeled by selective deprotonation of acidic groups. Conformational and dynamic changes were observed, consistent with those found in the crystal. Further, major solvent movement and NO escape were observed at pH 7, while the ligand remained in the heme binding pocket at pH 5. As a control, we also performed molecular dynamics (MD) simulations of sperm whale myoglobin, where NO migration into the interior cavities of the protein was observed, consistent with previous reports. We constructed a kinetic model of ligand escape to quantitatively relate the microscopic rate constants to the observed rates, and tested the predictions against the experimental data. The results suggest that release rates of diatomic molecules from heme proteins can be varied by several orders of magnitude through modest adjustments in geminate rebinding and gating behavior.

The contribution of heme propionate groups to the conformational dynamics associated with CO photodissociation from horse heart myoglobin

Photoacoustic calorimetry and transient absorption spectroscopy were used to study conformational dynamics associated with CO photodissociation from horse heart myoglobin (Mb) reconstituted with either Fe protoporphyrin IX dimethylester (FePPDME), Fe octaethylporphyrin (FeOEP), or with native Fe protoporphyrin IX (FePPIX). The volume and enthalpy changes associated with the Fe–CO bond dissociation and formation of a transient deoxyMb intermediate for the reconstituted Mbs were found to be similar to those determined for native Mb (Δ V 1 = −2.5 ± 0.6 ml mol −1 and Δ H 1 = 8.1 ± 3.0 kcal mol −1). The replacement of FePPIX by FeOEP significantly alters the conformational dynamics associated with CO release from protein. Ligand escape from FeOEP reconstituted Mb was determined to be roughly a factor of two faster ( τ = 330 ns) relative to native protein ( τ = 700 ns) and accompanying reaction volume and enthalpy changes were also found to be smaller (Δ V 2 = 5.4 ± 2.5 ml mol −1 and Δ H 2 = 0.7 ± 2.2 kcal mol −1) than those for native Mb (Δ V 2 = 14.3 ± 0.8 ml mol −1 and Δ H 2 = 7.8 ± 3.5 kcal mol −1). On the other hand, volume and enthalpy changes for CO release from FePPIX or FePPDME reconstituted Mb were nearly identical to those of the native protein. These results suggest that the hydrogen bonding network between heme propionate groups and nearby amino acid residues likely play an important role in regulating ligand diffusion through protein matrix. Disruption of this network leads to a partially open conformation of protein with less restricted ligand access to the heme binding pocket.

HTHP: A Novel Class of Hexameric, Tyrosine-coordinated Heme Proteins

We have cloned, expressed, isolated and characterized a hexameric tyrosine-coordinated heme protein (HTHP) from the marine bacterium Silicibacter pomeroyi. HTHP shows peroxidase and catalase activity and has a high thermal stability. As-isolated HTHP has absorption maxima at 407, 495, 504, 532 and 622 nm wavelength. Upon reduction maxima at 430, 564 and 596 nm wavelength are discernible. The crystal structure of HTHP reveals a hexameric, ring-like arrangement of six monomers. Each monomer binds a solvent accessible heme group, which is stabilized by the interaction of three neighboring monomers. The pocket around the heme distal side is positively charged due to three conserved arginine residues in direct vicinity. The heme iron is penta-coordinated with a tyrosine residue as proximal ligand. The coordinating hydroxyl-group of the tyrosine ligand interacts with the guanidinium group of a nearby arginine residue, an arrangement closely resembling the catalytic dyad found in monofunctional heme-containing catalases and coral allene oxide synthases, which are b-type cytochromes with tyrosine coordination trans to an empty coordination site. Despite the similarity in heme coordination HTHP is functionally and structurally unrelated to catalases and other heme-containing proteins. Its hexameric arrangement, solvent accessible heme binding pocket and heme coordination by tyrosine render HTHP a unique protein with unusual properties. A database search against complete and incomplete genomes shows that the 76 amino acid residues sequence of HTHP is unrelated to characterized proteins, but is homologous to orfs found in a phylogenetically diverse set of bacteria with sequence identities of 30–76%. We therefore propose that HTHP is the prototype of a new class of heme proteins.

Comparison of cytochromes b5 from insects and vertebrates

We report a 1.55 A X-ray crystal structure of the heme-binding domain of cytochrome b(5) from Musca domestica (house fly; HF b(5)), and compare it with previously published structures of the heme-binding domains of bovine microsomal cytochrome b(5) (bMc b(5)) and rat outer mitochondrial membrane cytochrome b(5) (rOM b(5)). The structural comparison was done in the context of amino acid sequences of all known homologues of the proteins under study. We show that insect b(5)s contain an extended hydrophobic patch at the base of the heme binding pocket, similar to the one previously shown to stabilize mammalian OM b(5)s relative to their Mc counterparts. The hydrophobic patch in insects includes a residue with a bulky hydrophobic side chain at position 71 (Met). Replacing Met71 in HF b(5) with Ser, the corresponding residue in all known mammalian Mc b(5)s, is found to substantially destabilize the holoprotein. The destabilization is a consequence of two related factors: (1) a large decrease in apoprotein stability and (2) extension of conformational disruption in the apoprotein beyond the empty heme binding pocket (core 1) and into the heme-independent folding core (core 2). Analogous changes have previously been shown to accompany replacement of Leu71 in rOM b(5) with Ser. That the stabilizing role of Met71 in HF b(5) is manifested primarily in the apo state is highlighted by the fact that its crystallographic Calpha B factor is modestly larger than that of Ser71 in bMc b(5), indicating that it slightly destabilizes local polypeptide conformation when heme is in its binding pocket. Finally, we show that the final unit of secondary structure in the cytochrome b(5) heme-binding domain, a 3(10) helix known as alpha6, differs substantially in length and packing interactions not only for different protein isoforms but also for given isoforms from different species.

Evolutionary trace analysis of plant haemoglobins: implications for site-directed mutagenesis

Haemoglobins are found ubiquitously in eukaryotes and many bacteria. In plants, haemoglobins were first identified in species, which can fix nitrogen via symbiosis with bacteria. Recent findings suggest that another class of haemoglobins termed as nonsymbiotic haemoglobins are present through out the plant kingdom and are expressed differentially during plant development. Limited data available suggests that non-symbiotic haemoglobins are involved in hypoxic stress and oversupply of nutrients. Due to lack of information on structurally conserved, functionally important residues in non-symbiotic haemoglobins, further studies to elucidate the molecular mechanisms underlying the biological role are hampered. To determine functionally important residues in non-symbiotic haemoglobins, I have analyzed a number of sequences from plant haemoglobin family, in the context of the known crystal structures of plant by evolutionary trace method. Results indicate that the, evolutionary trace method like conventional phylogentic analysis, could resolve phylogentic relationships between plant haemoglobin family. Evolutionary trace analysis has identified candidate functional (trace) residues that uniquely characterize the heme-binding pocket, dimer interface and possible novel functional surfaces. Such residues from specific three-dimensional clusters might be of functional importance in nonsymbiotic haemoglobins. These data, together with our improved knowledge of possible functional residues, can be used in future structure-function analysis experiments.

Characterization and kinetics studies of water buffalo (Bubalus bubalis) myoglobin

It is generally accepted that dry-aged buffalo (B. bubalis) meat becomes dark faster than bovine (B. taurus) meat, discouraging consumer purchase. We have investigated whether this faster darkening process might depend on structural and/or kinetic differences between buffalo and bovine myoglobins (Mbs). To this end, we have purified to homogeneity buffalo Mb from Longissimus dorsi muscle and obtained both its Mr (17,034.5) and the complete amino acid sequence, which, compared with the bovine one, showed three amino acid substitutions: Dbo141Ebu, Abo19Tbu and Abo117Dbu. As revealed by the 3D structure, they were located on the surface of the protein, far from the heme binding pocket, and did not cause appreciable structural changes. Autoxidation rates of purified buffalo and bovine myoglobins at 37 °C, pH 7.2, were almost identical (0.052±0.001 h−1 and 0.054±0.002 h−1, respectively), as were their oxygen-binding Kd values (3.7±0.1 μM and 3.5± 0.1 μM, respectively). These data indicate that the structure of the heme pockets in the two proteins is similar, with similar functional properties. Moreover, the percent of MetMb values in the purified buffalo and bovine samples, after the same time from slaughtering, were almost identical (57% and 47%, respectively). The results presented here suggest that the faster darkening of buffalo meat depends on factors other than the oxidation rate of its Mb, as, for example, the Mb content (0.393±0.005 g/100 g) and consequently its MetMb content, which is almost twice as high as bovine meat (0.209±0.003 g/100 g), and likely other factors.

Fungal Heme Oxygenases: Functional Expression and Characterization of Hmx1 from Saccharomyces cerevisiae and CaHmx1 from Candida albicans

Heme oxygenases convert heme to free iron, CO, and biliverdin. Saccharomyces cerevisiae and Candida albicans express putative heme oxygenases that are required for the acquisition of iron from heme, a critical process for fungal survival and virulence. The putative heme oxygenases Hmx1 and CaHmx1 from S. cerevisiae and C. albicans, respectively, minus the sequences coding for C-terminal membrane-binding domains, have been expressed in Escherichia coli. The C-terminal His-tagged, truncated enzymes are obtained as soluble, active proteins. Purified ferric Hmx1 and CaHmx1 have Soret absorption maxima at 404 and 410 nm, respectively. The apparent heme binding Kd values for Hmx1 and CaHmx1 are 0.34 +/- 0.09 microM and 1.0 +/- 0.2 microM, respectively. The resonance Raman spectra of Hmx1 reveal a heme binding pocket similar to those of the mammalian and bacterial heme oxygenases. Several reductants, including ascorbate, yeast cytochrome P450 reductase (CPR), human CPR, spinach ferredoxin/ferredoxin reductase, and putidaredoxin/putidaredoxin reductase, are able to provide electrons for biliverdin production by Hmx1 and CaHmx1. Of these, ascorbate is the most effective reducing partner. Heme oxidation by Hmx1 and CaHmx1 regiospecifically produces biliverdin IXalpha. Spectroscopic analysis of aerobic reactions with H2O2 identifies verdoheme as a reaction intermediate. Hmx1 and CaHmx1 are the first fungal heme oxygenases to be heterologously overexpressed and characterized. Their heme degradation activity is consistent with a role in iron acquisition.

Haem recognition by a Staphylococcus aureus NEAT domain

Successful pathogenic organisms have developed mechanisms to thrive under extreme levels of iron restriction. Haem-iron represents the largest iron reservoir in the human body and is a significant source of iron for some bacterial pathogens. NEAT (NEAr Transporter) domains are found exclusively in a family of cell surface proteins in Gram-positive bacteria. Many NEAT domain-containing proteins, including IsdA in Staphylococcus aureus, are implicated in haem binding. Here, we show that overexpression of IsdA in S. aureus enhances growth and an inactivation mutant of IsdA has a growth defect, compared with wild type, when grown in media containing haem as the sole iron source. Furthermore, the haem-binding property of IsdA is contained within the NEAT domain. Crystal structures of the apo-IsdA NEAT domain and in complex with haem were solved and reveal a clathrin adapter-like beta-sandwich fold with a large hydrophobic haem-binding pocket. Haem is bound with the propionate groups directed at the molecular surface and the iron is co-ordinated solely by Tyr(166). The phenol groups of Tyr(166) and Tyr(170) form an H-bond that may function in regulating haem binding and release. An analysis of IsdA structure-sequence alignments indicate that conservation of Tyr(166) is a predictor of haem binding by NEAT domains.

Characterization of Carbon Monoxide Photodissociation from FeIILPO with Photoacoustic Calorimetry

Lactoperoxidase belongs to a family of mammalian peroxidases that catalyze the oxidation of halides and small organic molecules in the presence of H2O2. We have used photoacoustic calorimetry to characterize thermodynamic parameters associated with ligand dissociation from bovine milk lactoperoxidase. Upon CO photorelease, a prompt (tau < 50 ns) exothermic volume contraction (DeltaH = -20 +/- 7 kcal mol-1 and DeltaH = -2 +/- 1 mL mol-1) was measured at pH 7.0 and 4.0, whereas an endothermic expansion (DeltaH = 30 +/- 13 kcal mol-1 and DeltaV = 9 +/- 2 mL mol(-1)) was observed at pH 10.0 and 7.0 in the presence of 500 mM NaCl. We attribute the observed volume and enthalpy changes to electrostriction arising from changes in the charge distribution associated with a reorganization of the heme binding pocket upon ligand dissociation. It is likely that cleavage of the Fe-CO bond is accompanied by distortion of a salt bridge between Arg557 and the heme propionate group, resulting in the observed electrostriction due to changes in charge distribution.

A Histidine/Tryptophan π-Stacking Interaction Stabilizes the Heme-Independent Folding Core of Microsomal Apocytochrome b5 Relative to that of Mitochondrial Apocytochrome b5,

The outer mitochondrial membrane isoform of mammalian cytochrome b5 (OM b5) is considerably more stable than its microsomal counterpart (Mc b5), whereas the corresponding apoproteins (OM and Mc apo-b5) exhibit similar stability. OM and Mc apo-b5 are also similar in that their empty heme-binding pockets (core 1) are highly disordered but that the remainder of each apoprotein (core 2) displays substantial hololike structure. Core 1 residue 71 is leucine in all known mammalian OM b5's and serine in the corresponding Mc proteins. Replacing Leu-71 in rat OM (rOM) b5 with Ser has been shown to (1) decrease apoprotein thermodynamic stability by >2 kcal/mol and (2) extend conformational disorder beyond core 1 and into core 2, as evidenced in part by loss of a near-UV circular dichroism signal associated with the side chain of invariant residue Trp-22. Herein we report identification of a conserved Mc b5 core 2 packing motif that plays a key role in stabilizing apoprotein conformation in the vicinity of Trp-22, thereby compensating for the presence of Ser at position 71: a pi-stacking interaction between the side chains of Trp-22 and His-15 that is extended by hydrogen bonding between the side chains of His-15, Ser-20, and Glu-11. The corresponding conserved packing motif in OM b5's differs in having arginine at position 15 and glutamate at position 20. We also present evidence indicating that the conserved Mc b5 packing motif noted above contributes to the unusually extensive secondary structure exhibited by bovine Mc apo-b5 in the urea-denatured state.

Characterization and kinetics studies of water buffalo ( Bubalus bubalis) myoglobin

The colour of buffalo ( Bubalus bubalis L.) meat is darker than bovine meat. Since meat colour depends on the concentration of myoglobin (Mb) and its oxidation state, we have determined the main structural and functional properties of buffalo Mb. Buffalo Mb was purified from longissimus dorsi muscles and its molecular mass determined by ESI Q-TOF mass spectrometry. The molecular mass 17,034.50 was 86.20 Da higher than the bovine Mb. This was confirmed by analysing its primary structure, using a combined approach based on Edman degradation and MALDI-TOF mass spectrometry. Comparing the amino acid sequences of both Mbs, we found three amino acid differences out of 153 amino acid residues. One is a conservative substitution (D bov141E buf) , and the other two (A bov19T buf and A bov117D buf) are nonconservative. These amino acid substitutions are unlikely to cause structural changes because they are located far from the heme binding pocket, as revealed by the 3D structure of buffalo Mb elaborated by homology modelling. Stability analyses show no difference with the bovine Mb for helix E and only minor differences in the stability values for helices A and G. Moreover, autoxidation rates of purified buffalo and bovine myoglobins at 37 °C, pH 7.2, were almost identical, 0.052 ± 0.001 h − 1 and 0.054 ± 0.002 h − 1 , respectively, as were their oxygen-binding Kd values, 3.7 ± 0.1 μM and 3.5 ± 0.1 μM, respectively. The percent of MetMb values were almost identical. The results presented here suggest that the darker buffalo meat depends on factors other than the oxidation rate of its Mb, as, for example, the Mb content (0.393 ± 0.005 g/100 g of tissue) and consequently MetMb, which are almost twice as high as bovine meat (Mb: 0.209 ± 0.003 g/100 g of tissue).

Identification of residues crucially involved in soluble guanylate cyclase activation

The ubiquitous heterodimeric nitric oxide (NO) receptor soluble guanylate cyclase (sGC) plays a key role in various signal transduction pathways. Binding of NO takes place at the prosthetic heme moiety at the N-terminus of the β 1-subunit of sGC. The induced structural changes lead to an activation of the catalytic C-terminal domain of the enzyme and to an increased conversion of GTP into the second messenger cyclic GMP (cGMP). In the present work we selected and substituted different residues of the sGC heme-binding pocket based on a sGC homology model. The generated sGC variants were tested in a cGMP reporter cell for their effect on the enzyme activation by heme-dependent (NO, BAY 41-2272) stimulators and heme-independent (BAY 58-2667) activators. The use of these experimental tools allows the enzyme’s heme content to be explored in a non-invasive manner. Asp 44, Asp 45 and Phe 74 of the β 1-subunit were identified as being crucially important for functional enzyme activation. β 1Asp 45 may serve as a switch between different conformational states of sGC and point to a possible mechanism of action of the heme dependent sGC stimulator BAY 41-2272. Furthermore, our data shows that the activation profile of β 1IIe 145 Tyr is unchanged compared to the native enzyme, suggesting that Tyr 145 does not confer the ability to distinguish between NO and O 2. In summary, the present work further elucidated intramolecular mechanisms underlying the NO- and BAY 41-2272-mediated sGC activation and raises questions regarding the postulated role of Tyr 145 for ligand discrimination.