Heme Prosthetic Group Research Articles

Crystal structure and statistical coupling analysis of highly glycosylated peroxidase from royal palm tree (Roystonea regia)

Royal palm tree peroxidase (RPTP) is a very stable enzyme in regards to acidity, temperature, H(2)O(2), and organic solvents. Thus, RPTP is a promising candidate for developing H(2)O(2)-sensitive biosensors for diverse applications in industry and analytical chemistry. RPTP belongs to the family of class III secretory plant peroxidases, which include horseradish peroxidase isozyme C, soybean and peanut peroxidases. Here we report the X-ray structure of native RPTP isolated from royal palm tree (Roystonea regia) refined to a resolution of 1.85A. RPTP has the same overall folding pattern of the plant peroxidase superfamily, and it contains one heme group and two calcium-binding sites in similar locations. The three-dimensional structure of RPTP was solved for a hydroperoxide complex state, and it revealed a bound 2-(N-morpholino) ethanesulfonic acid molecule (MES) positioned at a putative substrate-binding secondary site. Nine N-glycosylation sites are clearly defined in the RPTP electron-density maps, revealing for the first time conformations of the glycan chains of this highly glycosylated enzyme. Furthermore, statistical coupling analysis (SCA) of the plant peroxidase superfamily was performed. This sequence-based method identified a set of evolutionarily conserved sites that mapped to regions surrounding the heme prosthetic group. The SCA matrix also predicted a set of energetically coupled residues that are involved in the maintenance of the structural folding of plant peroxidases. The combination of crystallographic data and SCA analysis provides information about the key structural elements that could contribute to explaining the unique stability of RPTP.

Ionic-liquid/NH 2-MWCNTs as a highly sensitive nano-composite for catalase direct electrochemistry

A nano-composite material consisting of amine functionalized multi-walled carbon nanotubes and a room temperature ionic-liquid (1-butyl-3-methylimidazolium tetrafluoroborate) was prepared and used to construct a novel catalase (Ct) based biosensor for the determination of hydrogen peroxide. The modified electrode exhibited a quasi-reversible cyclic voltammogram corresponding to the Fe(II)/Fe(III) redox couple in the heme prosthetic group of Ct with a formal potential of −460 mV in 0.1 M phosphate buffer solution at pH 7.0. The nano-composite film showed an obvious promotion of the direct electron transfer between Ct and the underlying electrode. The apparent charge transfer rate constant and transfer coefficient for electron transfer between the electrode surface and enzyme were calculated as 2.23 s −1 and 0.45, respectively. The immobilized Ct exhibited a relatively high sensitivity (4.9 nA/nM) toward hydrogen peroxide. Under the optimized experimental conditions, hydrogen peroxide was detected in the concentration range from 8.6 to 140 nM with a detection limit of 3.7 nM at S/N = 3. The modified electrode was stable for two weeks with no observable change in the cyclic voltammograms.

Heme oxygenase-1 affords protection against noncerebral forms of severe malaria

Infection by Plasmodium, the causative agent of malaria, is associated with hemolysis and therefore with release of hemoglobin from RBC. Under inflammatory conditions, cell-free hemoglobin can be oxidized, releasing its heme prosthetic groups and producing deleterious free heme. Here we demonstrate that survival of a Plasmodium-infected host relies strictly on its ability to prevent the cytotoxic effects of free heme via the expression of the heme-catabolyzing enzyme heme oxygenase-1 (HO-1; encoded by the Hmox1 gene). When infected with Plasmodium chabaudi chabaudi (Pcc), wild-type (Hmox1(+/+)) BALB/c mice resolved infection and restored homeostasis thereafter (0% lethality). In contrast, HO-1 deficient (Hmox1(-/-)) BALB/c mice developed a lethal form of hepatic failure (100% lethality), similar to the one occurring in Pcc-infected DBA/2 mice (75% lethality). Expression of HO-1 suppresses the pro-oxidant effects of free heme, preventing it from sensitizing hepatocytes to undergo TNF-mediated programmed cell death by apoptosis. This cytoprotective effect, which inhibits the development of hepatic failure in Pcc-infected mice without interfering with pathogen burden, is mimicked by pharmacological antioxidants such as N-acetylcysteine (NAC). When administered therapeutically, i.e., after Pcc infection, NAC suppressed the development of hepatic failure in Pcc-infected DBA/2 mice (0% lethality), without interfering with pathogen burden. In conclusion, we describe a mechanism of host defense against Plasmodium infection, based on tissue cytoprotection against free heme and limiting disease severity irrespectively of parasite burden.

Novel Mutations inCYP11B1Gene Leading to 11β-Hydroxylase Deficiency in Brazilian Patients

Deficiency of 11 beta-hydroxylase results in the impairment of the last step of cortisol synthesis. In females, the phenotype of this disorder includes different degrees of genital ambiguity and arterial hypertension. Mutations in the CYP11B1 gene are responsible for this disease.The objective of the study was to screen the CYP11B1 gene for mutations in two unrelated Brazilian females with congenital adrenal hyperplasia due to 11 beta-hydroxylase deficiency.The coding and intron-exon junction regions of CYP11B1 were totally sequenced. A putative splice mutation was further investigated by minigene transcription.We report two novel CYP11B1 mutations in these Brazilian patients. An Arabian Lebanese descendent female was found to be homozygous for a cytosine insertion at the beginning of exon 8, changing the 404 arginine to proline. It alters the open reading frame, creating a putative truncated protein at 421 residue, which eliminates the domain necessary for the association of heme prosthetic group. A severely virilized female was homozygous for the g.2791G>A transition in the last position of exon 4. This nucleotide is also part of 5' intron 4 donor splice site consensus sequence. Minigene experiments demonstrated that g.2791G>A activated an alternative splice site within exon 4, leading to a 45-bp deletion in the transcript. The putative translation of such modified mRNA indicates a truncated protein at residue 280.We describe two novel mutations, g.4671_4672insC and g.2791G>A, that drastically affects normal protein structure. These mutations abolish normal enzyme activity, leading to a severe phenotype of congenital adrenal hyperplasia due to 11 beta-hydroxylase deficiency.

Hydrogen/Deuterium Exchange Mass Spectrometry with Top-Down Electron Capture Dissociation for Characterizing Structural Transitions of a 17 kDa Protein

Amide H/D exchange (HDX) mass spectrometry (MS) is widely used for protein structural studies. Traditionally, this technique involves protein labeling in D(2)O, followed by acid quenching, proteolytic digestion, and analysis of peptide deuteration levels by HPLC/MS. There is great interest in the development of alternative HDX approaches involving the top-down fragmentation of electrosprayed protein ions, instead of relying on enzymatic cleavage and solution-phase separations. A number of recent studies have demonstrated that electron capture dissociation (ECD) results in fragmentation of gaseous protein ions with little or no H/D scrambling. However, the successful application of this approach for in-depth protein conformational studies has not yet been demonstrated. The current work uses horse myoglobin as a model system for assessing the suitability of HDX-MS with top-down ECD for experiments of this kind. It is found that ECD can pinpoint the locations of protected amides with an average resolution of less than two residues for this 17 kDa protein. Native holo-myoglobin (hMb) shows considerable protection from exchange in all of its helices, whereas loops are extensively deuterated. Fraying is observable at some helix termini. Removal of the prosthetic heme group from hMb produces apo-myoglobin (aMb). Both hMb and aMb share virtually the same HDX protection pattern in helices A-E, whereas helix F is unfolded in aMb. In addition, destabilization is evident for some residues close to the beginning of helix G, the end of helix H, and the C-terminus of the protein. The structural changes reported herein are largely consistent with earlier NMR data for sperm whale myoglobin, although small differences between the two systems are evident. Our findings demonstrate that the level of structural information obtainable with top-down ECD for small to medium-sized proteins considerably surpasses that of traditional HDX-MS experiments, while at the same time greatly reducing undesired amide back exchange.

The Discovery and Characterization of Molybdopterin: the Work of K. V. Rajagopalan

Hepatic Sulfite Oxidase. A Functional Role for Molybdenum (Cohen, H. J., Fridovich, I., and Rajagopalan, K. V. (1971) J. Biol. Chem. 246, 374–382) Characterization of the Molybdenum Cofactor of Sulfite Oxidase, Xanthine, Oxidase, and Nitrate Reductase. Identification of a Pteridine as a Structural Component (Johnson, J. L., Hainline, B. E., and Rajagopalan, K. V. (1980) J. Biol. Chem. 255, 1783–1786) The Structure of the Molybdenum Cofactor. Characterization of Di(carboxamidomethyl)molybdopterin from Sulfite Oxidase and Xanthine Oxidase (Kramer, S. P., Johnson, J. L., Ribeiro, A. A., Millington, D. S., and Rajagopalan, K. V. (1987) J. Biol. Chem. 262, 16357–16363)

Heme- a, the heme prosthetic group of cytochrome c oxidase, is increased in Alzheimer's disease

Heme- a, is the heme prosthetic group of cytochrome c oxidase (COX), the terminal complex of the mitochondrial electron transport chain. We measured heme- a levels in postmortem brain tissue from nine patients diagnosed with dementia: Alzheimer's disease (AD) was the primary diagnosis in five, AD/diffuse Lewy body disease (DLBD) was diagnosed in two, DLBD was diagnosed in one, and DLBD (severe)/AD (mild) was diagnosed in one. Eight non-demented patients who died from non-neurological causes served as controls. When the primary diagnosis was AD (AD and AD/DLBD), levels of cerebral heme- a were increased almost two-fold on a protein basis compared to controls ( p < 0.001). Using perfused and non-perfused rats we showed that measured levels of cerebral heme- a were unaffected by the presence of blood in brain tissue. In mice we showed that levels of cerebral heme- a were unaffected by 24 h of storage at 4 °C prior to freezing. These animal studies suggest that increased levels of cerebral heme- a in AD were not due to blood in postmortem brain or variation in postmortem interval.

Soluble Guanylate Cyclase

Arthur Kornberg, who shared with Severo Ochoa the 1959 Nobel Prize for Physiology or Medicine, stated in his autobiography that he had never met a dull enzyme.1 We strongly believe that soluble guanylate cyclase (sGC; E.C.4.6.1.2) is no exception to this. Article see p 2781 Nitric oxide (NO) plays an important physiological role as a signaling molecule as well as a cytotoxic agent. It is produced by 3 distinct NO synthases, specifically endothelial, neuronal, and inducible NO synthase. In the vasculature, endothelial NO synthase plays an important role in vascular homeostasis because NO generated in endothelial cells promotes vascular smooth muscle cell relaxation and inhibits platelet aggregation. Importantly, bioavailability of NO can be reduced in cardiovascular diseases, a condition which has been termed “endothelial dysfunction.” This phenomenon provides a rationale for therapeutic intervention. The primary target of NO is sGC, which is a heterodimeric enzyme consisting of an α- and β-subunit and a prosthetic heme group, which is located in the β-subunit. The heme group contains a ferrous iron atom (Fe2+). Binding of NO to this iron changes the conformation of the enzyme and leads to a >100-fold increase in catalytic rate (ie, the conversion of guanosine triphosphate to the second messenger cyclic guanosine monophosphate [cGMP]). The NO-induced cGMP signal modulates intracellularly the activity of several effector molecules: cGMP-dependent protein kinases, cGMP-regulated phosphodiesterases, and cGMP-gated ion channels. Of note, both heme iron oxidation (Fe3+) and heme removal render sGC unresponsive to NO (Figure). These 2 conditions could therefore provide a mechanism for “vascular smooth muscle cell dysfunction” in cardiovascular disease states, which are often characterized by marked vasoconstriction either systemically or in specific organs. Figure. Schematic illustrating 3 different forms of soluble guanylate cyclase and their respective responsiveness to nitrovasodilators, sGC stimulators (eg, BAY 41–2272), and sGC …

Benzylic Oxidation of Gemfibrozil-1-O-β-Glucuronide by P450 2C8 Leads to Heme Alkylation and Irreversible Inhibition

Gemfibrozil-1-O-beta-glucuronide (GEM-1-O-gluc), a major metabolite of the antihyperlipidemic drug gemfibrozil, is a mechanism-based inhibitor of P450 2C8 in vitro, and this irreversible inactivation may lead to clinical drug-drug interactions between gemfibrozil and other P450 2C8 substrates. In light of this in vitro finding and the observation that the glucuronide conjugate does not contain any obvious structural alerts, the current study was conducted to determine the potential site of GEM-1-O-gluc bioactivation and the subsequent mechanism of P450 2C8 inhibition (i.e., modification of apoprotein or heme). LC/MS analysis of a reaction mixture containing recombinant P450 2C8 and GEM-1-O-gluc revealed that the substrate was covalently linked to the heme prosthetic heme group during catalysis. A combination of mass spectrometry and deuterium isotope effects revealed that a benzylic carbon on the 2',5'-dimethylphenoxy group of GEM-1-O-gluc was covalently bound to the heme of P450 2C8. The regiospecificity of substrate addition to the heme group was not confirmed experimentally, but computational modeling experiments indicated that the gamma-meso position was the most likely site of modification. The metabolite profile, which consisted of two benzyl alcohol metabolites and a 4'-hydroxy-GEM-1-O-gluc metabolite, indicated that oxidation of GEM-1-O-gluc was limited to the 2',5'-dimethylphenoxy group. These results are consistent with an inactivation mechanism wherein GEM-1-O-gluc is oxidized to a benzyl radical intermediate, which evades oxygen rebound, and adds to the gamma-meso position of heme. Mechanism-based inhibition of P450 2C8 can be rationalized by the formation of the GEM-1-O-gluc-heme adduct and the consequential restriction of additional substrate access to the catalytic iron center.

Evidence for Heme Oxygenase Activity in a Heme Peroxidase

The heme peroxidase and heme oxygenase enzymes share a common heme prosthetic group but catalyze fundamentally different reactions, the first being H(2)O(2)-dependent oxidation of substrate using an oxidized Compound I intermediate, and the second O(2)-dependent degradation of heme. It has been proposed that these enzymes utilize a common reaction intermediate, a ferric hydroperoxide species, that sits at a crossroads in the mechanism and beyond which there are two mutually exclusive mechanistic pathways. Here, we present evidence to support this proposal in a heme peroxidase. Hence, we describe kinetic data for a variant of ascorbate peroxidase (W41A) which reacts slowly with tert-butyl hydroperoxide and does not form the usual peroxidase Compound I intermediate; instead, structural data show that a product is formed in which the heme has been cleaved at the alpha-meso position, analogous to the heme oxygenase mechanism. We interpret this to mean that the Compound I (peroxidase) pathway is shut down, so that instead the reaction intermediate diverts through the alternative (heme oxygenase) route. A mechanism for formation of the product is proposed and discussed in the light of what is known about the heme oxygenase reaction mechanism.

Folding energy landscape of cytochrome cb 562

Cytochrome cb(562) is a variant of an Escherichia coli four-helix bundle b-type heme protein in which the porphyrin prosthetic group is covalently ligated to the polypeptide near the terminus of helix 4. Studies from other laboratories have shown that the apoprotein folds rapidly without the formation of intermediates, whereas the holoprotein loses heme before native structure can be attained. Time-resolved fluorescence energy transfer (TRFET) measurements of cytochrome cb(562) refolding triggered using an ultrafast continuous-flow mixer (150 micros dead time) reveal that heme attachment to the polypeptide does not interfere with rapid formation of the native structure. Analyses of the TRFET data produce distributions of Trp-59-heme distances in the protein before, during, and after refolding. Characterization of the moments and time evolution of these distributions provides compelling evidence for a refolding mechanism that does not involve significant populations of intermediates. These observations suggest that the cytochrome b(562) folding energy landscape is minimally frustrated and able to tolerate the introduction of substantial perturbations (i.e., the heme prosthetic group) without the formation of deep misfolded traps.

Tetrahydrobiopterin Recycling, a Key Determinant of Endothelial Nitric-oxide Synthase-dependent Signaling Pathways in Cultured Vascular Endothelial Cells

Tetrahydrobiopterin (BH4) is a key redox-active cofactor in endothelial isoform of NO synthase (eNOS) catalysis and is an important determinant of NO-dependent signaling pathways. BH4 oxidation is observed in vascular cells in the setting of the oxidative stress associated with diabetes. However, the relative roles of de novo BH4 synthesis and BH4 redox recycling in the regulation of eNOS bioactivity remain incompletely defined. We used small interference RNA (siRNA)-mediated "knockdown" GTP cyclohydrolase-1 (GTPCH1), the rate-limiting enzyme in BH4 biosynthesis, and dihydrofolate reductase (DHFR), an enzyme-recycling oxidized BH4 (7,8-dihydrobiopterin (BH2)), and studied the effects on eNOS regulation and biopterin metabolism in cultured aortic endothelial cells. Knockdown of either DHFR or GTPCH1 attenuated vascular endothelial growth factor (VEGF)-induced eNOS activity and NO production; these effects were recovered by supplementation with BH4. In contrast, supplementation with BH2 abolished VEGF-induced NO production. DHFR but not GTPCH1 knockdown increased reactive oxygen species (ROS) production. The increase in ROS production seen with siRNA-mediated DHFR knockdown was abolished either by simultaneous siRNA-mediated knockdown of eNOS or by supplementing with BH4. In contrast, addition of BH2 increased ROS production; this effect of BH2 was blocked by BH4 supplementation. DHFR but not GTPCH1 knockdown inhibited VEGF-induced dephosphorylation of eNOS at the inhibitory site serine 116; these effects were recovered by supplementation with BH4. These studies demonstrate a striking contrast in the pattern of eNOS regulation seen by the selective modulation of BH4 salvage/reduction versus de novo BH4 synthetic pathways. Our findings suggest that the depletion of BH4 is not sufficient to perturb NO signaling, but rather that concentration of intracellular BH2, as well as the relative concentrations of BH4 and BH2, together play a determining role in the redox regulation of eNOS-modulated endothelial responses.

Global and Local Structural Changes of Protein Unfolding

AbstractSummery: In the mean‐field Ising model, the folding‐unfolding behavior of a protein is regarded as an ensemble of units reduced from, presumably, peptide bonds or amino acid residues. Units of similar thermodynamic properties are further classified into groups that are related to, for instance, α‐helices or β‐sheets. Units of the same group are assumed to unfold collectively, whereas those of different groups may undergo either sequential or coupled unfolding. The introduction of unfolding groups facilitates the description of non‐collective local structural changes experimentally observed via a multi‐group unfolding. We incorporate denaturants and temperature effects into the free energy expression of the protein upon dissolution in a specific environment at thermal equilibrium, and a model protein, cytochrome c, was examined. Results indicate that there are at least four unfolding groups induced unfolding of cytochrome c: two are related to the prosthetic heme group, whereas another two groups are α‐helices and global nearly group, which largely account for global changes in protein morphology. The extracted thermodynamic parameters, on the basis of the Ising model, can closely predict unfolding behaviors of the proteins in compounded denaturing environments.

Different Modes of Binding of Mono-, Di-, and Trihalogenated Phenols to the Hemoglobin Dehaloperoxidase from Amphitrite ornata

The hemoglobin dehaloperoxidase (DHP), found in the coelom of the terebellid polychaete Amphitrite ornata, is a dual-function protein that has the characteristics of both hemoglobins and peroxidases. In addition to oxygen transport function, DHP readily oxidizes halogenated phenols in the presence of hydrogen peroxide. The peroxidase activity of DHP is high relative to that of wild-type myoglobin or hemoglobin, but the most definitive difference in DHP is a well-defined substrate-binding site in the distal pocket, which was reported for 4-iodophenol in the X-ray crystal structure of DHP. The binding of 2,4,6-trihalogenated phenols is relevant since 2,4,6-tribromophenol is considered to be the native substrate and 2,4,6-trichlorophenol also gives high turnover rates in enzymatic studies. The most soluble trihalogenated phenol, 2,4,6-trifluorophenol, acts as a highly soluble structural analogue to the native substrate 2,4,6-tribromophenol. To improve our understanding of substrate binding, we compared the most soluble substrate analogues, 4-bromophenol, 2,4-dichlorophenol, and 2,4,6-trifluorophenol, using (1)H and (19)F NMR to probe substrate binding interactions in the active site of the low-spin metcyano adduct of DHP. Both mono- and dihalogenated phenols induced changes in resonances of the heme prosthetic group and an internal heme edge side chain, while (1)H NMR, (19)F NMR, and relaxation data for a 2,4,6-trihalogenated substrate indicate a mode of binding on the exterior of DHP. The differences in binding are correlated with differences in enzymatic activity for the substrates studied.

Determination of Cytochrome c and Other Heme Proteins Using the Reduction Wave of Mercury Protoporphyrin IX Groups Generated by a Hydroxylamine Induced Replacement Reaction

We have found that in the presence of hydroxylamine, the heme prosthetic group of the heme protein adsorbed at the mercury electrode surface reacts with mercury ion produced by the electrochemical oxidation of mercury and is quantitatively converted into the mercury protoporphyrin IX group using single-sweep polarography. As a result, the small redox peak P(0) of the heme prosthetic group at about -0.46 V (vs SCE) disappears and a large new reduction peak P of mercury protoporphyrin IX group at -0.89 V comes out in a pH 9.6 NaHCO(3)-Na(2)CO(3) solution. Peak P is extremely sensitive to heme protein concentration. On the basis of the reduction peak P, a unique electrochemical method for heme protein assays is constructed. For the cytochrome c determination, the peak height is linearly proportional to the concentration in the range of 0.005-15 mg L(-1) (correlation coefficient 0.999). The detection limit is 0.003 mg L(-1). In contrast with peak P(0), the detection limit of cytochrome c is only 0.6 mg L(-1). The voltammograms of heme proteins in the absence and presence of hydroxylamine can serve as a reliable qualitative analytical method. The chemical reaction is peculiar to the heme prosthetic group. Without hydroxylamine it cannot occur. Thereby the method is highly specific and free from interference. The performance takes only a few minutes. These advantages make the method attractive for heme protein detecting.

Surface Modification Affects the Heme Planarity and Accessibility in Horseradish Peroxidase

Some noncovalently linked hemes like those in the peroxidases have highly conserved characteristic distortions in the porphyrin plane. Conservation occurs even for some proteins with a large natural variation in the amino acid sequence. Thus it is reasonable to anticipate that nonplanar porphyrins and protein-induced changes in the planarity may provide a mechanism for protein modulation of biological properties.We previously reported that covalent modification of three accessible charged lysines (Lys-174, Lys-232, Lys-241) to the hydrophobic anthraquinolysine residues successfully improves electron transfer properties, catalytic efficiency, and stability of HRP.Here we report that this modification also alters the Heme planarity and accessibility. Using the known x-ray crystallographic structure of HRP, two 3D models of HRP representing the native and modified enzyme, were constructed. Molecular dynamics simulations showed that upon modification, the accessibility of the heme prosthetic group to the substrates increased from 39.6 to 99.7 A2. Moreover, the Heme Planarity also affected significantly. While in the native structure the heme plane is concave and curves toward the proximal His, in the modified enzyme the heme assumes a planar conformation and shifts the position of the proximal His. This change causes the heme iron to become more exposed on the distal side of the porphyrin plane and so more accessible to the peroxide substrate. This result agrees well with the experimentally observed enhanced reactivity of the modified enzyme to the peroxide substrates.

Changes in oxyhemoglobin during emotional oddball task in healthy subject evaluation multi channel NIRS

Here, the effect of benzene on hemoglobin structure, stability and heme prosthetic group integrity was studied by different methods. These included UV–vis absorption spectrophotometry, normal and synchronous fluorescence techniques, and differential scanning calorimetry (DSC). Our results indicated that benzene has high hemolytic potential even at low concentrations. The UV–vis spectroscopic results demonstrated that benzene altered both the globin chain and the heme prosthetic group of hemoglobin increasing met- and deoxy-Hb, while decreasing oxy-Hb. However, with increasing benzene the concentration of all species decreased due to heme destruction. The spectrophotometric results show that benzene has a high potential for penetrating the hydrophobic pocket of hemoglobin. These results were consistent with the molecular docking simulation results of benzene-hHb. Aggregation and thermal denaturation studies show that the increased benzene concentration induced hemoglobin aggregation with a decrease in stability, which is consistent with the DSC results. Conventional fluorescence spectroscopy revealed that the heme degradation species were produced in the presence of benzene. The results of constant wavelength synchronous fluorescence spectroscopy (CWSFS) indicated that at least five heme-degraded species were produced. Together, our results indicated that benzene has adverse effects on hemoglobin structure and function, and heme degradation.

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Probing the Function of Heme Distortion in the H-NOX Family

Hemoproteins carry out diverse functions utilizing a wide range of chemical reactivity while employing the same heme prosthetic group. It is clear from high-resolution crystal structures and biochemical studies that protein-bound hemes are not planar and adopt diverse conformations. The crystal structure of an H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX) contains the most distorted heme reported to date. In this study, Tt H-NOX was engineered to adopt a flatter heme by mutating proline 115, a conserved residue in the H-NOX family, to alanine. Decreasing heme distortion in Tt H-NOX increases affinity for oxygen and decreases the reduction potential of the heme iron. Additionally, flattening the heme is associated with significant shifts in the N-terminus of the protein. These results show a clear link between the heme conformation and Tt H-NOX structure and demonstrate that heme distortion is an important determinant for maintaining biochemical properties in H-NOX proteins.

Defining the in Vivo Role for Cytochrome b5 in Cytochrome P450 Function through the Conditional Hepatic Deletion of Microsomal Cytochrome b5

In vitro, cytochrome b5 modulates the rate of cytochrome P450-dependent mono-oxygenation reactions. However, the role of this enzyme in determining drug pharmacokinetics in vivo and the consequential effects on drug absorption distribution, metabolism, excretion, and toxicity are unclear. In order to resolve this issue, we have carried out the conditional deletion of microsomal cytochrome b5 in the liver to create the hepatic microsomal cytochrome b5 null mouse. These mice develop and breed normally and have no overt phenotype. In vitro studies using a range of substrates for different P450 enzymes showed that in hepatic microsomal cytochrome b5 null NADH-mediated metabolism was essentially abolished for most substrates, and the NADPH-dependent metabolism of many substrates was reduced by 50–90%. This reduction in metabolism was also reflected in the in vivo elimination profiles of several drugs, including midazolam, metoprolol, and tolbutamide. In the case of chlorzoxazone, elimination was essentially unchanged. For some drugs, the pharmacokinetics were also markedly altered; for example, when administered orally, the maximum plasma concentration for midazolam was increased by 2.5-fold, and the clearance decreased by 3.6-fold in hepatic microsomal cytochrome b5 null mice. These data indicate that microsomal cytochrome b5 can play a major role in the in vivo metabolism of certain drugs and chemicals but in a P450- and substrate-dependent manner.