Biomedical subjects

T L Poulos

Publications and source records attributed to T L Poulos.

At least 37 records · Page 2Linked to original sources

PubMed1999

Fatty acid metabolism, conformational change, and electron transfer in cytochrome P-450(BM-3).

Crystal structure-based mutagenesis studies on cytochrome P-450(BM-3) have confirmed the importance of R47, Y51, and F87 in substrate binding. Replacing F87 has profound effects on regioselectivity. In contrast, changing either R47 or Y51 alone to other residues results in limited impact on substrate binding affinity. Mutating both, however, leads to large changes. Substrate-induced protein conformational changes not only lead to specific substrate binding in the heme domain, but also affect interactions with the FMN domain. Unlike the microsomal P-450 reductase, the FMN semiquinone is the active electron donor to the heme iron in P-450(BM-3). The crystal structure of P-450(BM-3) heme/FMN bidomain provides important insights into why the FMN semiquinone is the preferred electron donor to the heme as well as how substrate-induced structural changes possibly affect the FMN and heme domain-domain interaction.

Bacillus megaterium↗

PubMed1999

Crystal structures of zinc-free and -bound heme domain of human inducible nitric-oxide synthase. Implications for dimer stability and comparison with endothelial nitric-oxide synthase.

The crystal structures of the heme domain of human inducible nitric-oxide synthase (NOS-2) in zinc-free and -bound states have been solved. In the zinc-free structure, two symmetry-related cysteine residues form a disulfide bond. In the zinc-bound state, these same two cysteine residues form part of a zinc-tetrathiolate (ZnS(4)) center indistinguishable from that observed in the endothelial isoform (NOS-3). As in NOS-3, ZnS(4) plays a key role in stabilizing intersubunit contacts and in maintaining the integrity of the cofactor (tetrahydrobiopterin) binding site of NOS-2. A comparison of NOS-2 and NOS-3 structures illustrates the conservation of quaternary structure, tertiary topology, and substrate and cofactor binding sites, in addition to providing insights on isoform-specific inhibitor design. The structural comparison also reveals that pterin binding does not preferentially stabilize the dimer interface of NOS-2 over NOS-3.

Dimerization↗

PubMed1999

The effects of an engineered cation site on the structure, activity, and EPR properties of cytochrome c peroxidase.

Earlier work [Bonagura et al. (1996) Biochemistry 35, 6107] showed that the K+ site found in the proximal pocket of ascorbate peroxidase (APX) could be engineered into cytochrome c peroxidase (CCP). Binding of K+ at the engineered site results in a loss in activity and destabilization of the CCP compound I Trp191 cationic radical owing to long-range electrostatic effects. The engineered CCP mutant crystal structure has been refined to 1.5 A using data obtained at cryogenic temperatures which provides a more detailed basis for comparison with the naturally occurring K+ site in APX. The characteristic EPR signal associated with the Trp191 radical becomes progressively weaker as K+ is added, which correlates well with the loss in enzyme activity as [K+] is increased. These results coupled with stopped-flow studies support our earlier conclusions that the loss in activity and EPR signal is due to destabilization of the Trp191 cationic radical.

Binding Sites↗

PubMed1999

Structure of a cytochrome P450-redox partner electron-transfer complex.

The crystal structure of the complex between the heme- and FMN-binding domains of bacterial cytochrome P450BM-3, a prototype for the complex between eukaryotic microsomal P450s and P450 reductase, has been determined at 2.03 A resolution. The flavodoxin-like flavin domain is positioned at the proximal face of the heme domain with the FMN 4.0 and 18.4 A from the peptide that precedes the heme-binding loop and the heme iron, respectively. The heme-binding peptide represents the most efficient and coupled through-bond electron pathway to the heme iron. Substantial differences between the FMN-binding domains of P450BM-3 and microsomal P450 reductase, observed around the flavin-binding sites, are responsible for different redox properties of the FMN, which, in turn, control electron flow to the P450.

Amino Acid Sequence↗

PubMed1999

A study of the K(+)-site mutant of ascorbate peroxidase: mutations of protein residues on the proximal side of the heme cause changes in iron ligation on the distal side.

A series of ferric and ferrous derivatives of wild-type ascorbate peroxidase (APX) and of an engineered K(+)-site mutant of APX that has had its potassium cation binding site removed have been examined by electronic absorption and magnetic circular dichroism (MCD) spectroscopy at 4 degrees C. Wild-type ferric APX has spectroscopic properties that are very similar to those of ferric cytochrome c peroxidase (CCP) and likely exists primarily as a five-coordinate high-spin heme ligated on the proximal side by a histidine at pH 7. There is also evidence for minority contributions from six-coordinate high- and low-spin species (histidine-water, histidine-hydroxide, and bis-histidine). The K(+)-site mutant of APX varies considerably in the electronic absorption and MCD spectra in both the ferric and ferrous states when compared with spectra of the wild-type APX. The electronic absorption and MCD spectra of the engineered K(+)-site APX mutant are essentially identical to those of cytochrome b5, a known bis-imidazole (histidine) ligated heme system. It therefore appears that the K(+)-site mutant of APX has undergone a conformational change to yield a bis-histidine coordination structure in both the ferric and ferrous oxidation states at neutral pH. This conformational change is the result of mutagenesis of the protein to remove the K(+)-binding site which is located approximately 8 A from the peroxide binding pocket. Thus, mutations of protein residues on the proximal side of the heme cause changes in iron ligation on the distal side.

Ascorbate Peroxidases↗

PubMed1999

Heme-mediated oxygen activation in biology: cytochrome c oxidase and nitric oxide synthase.

Major advances have been made in our understanding of cytochrome c oxidase owing to continued crystallographic work on important intermediates. This, together with a wealth of data derived from selective mutations and sophisticated spectroscopic probes, has provided significant new insights into oxidase dioxygen chemistry and proton pumping activities. Recent advances have also been made for nitric oxide synthase, owing to the crystal structure determination of the heme domain for two of three nitric oxide synthase isoforms.

Animals↗

PubMed1999

Helping copper find a home.

The crystal structure of the superoxide dismutase copper chaperone provides some key insights into the molecular mechanism of copper trafficking.

Biological Transport↗

PubMed1999

Crystal structure of human heme oxygenase-1.

Heme oxygenase catalyzes the first step in the oxidative degradation of heme. The crystal structure of heme oxygenase-1 (HO-1) reported here reveals a novel helical fold with the heme sandwiched between two helices. The proximal helix provides a heme iron ligand, His 25. Conserved glycines in the distal helix near the oxygen binding site allow close contact between the helix backbone and heme in addition to providing flexibility for substrate binding and product release. Regioselective oxygenation of the alpha-meso heme carbon is due primarily to steric influence of the distal helix.

Binding Sites↗

PubMed1998

Crystal structure of constitutive endothelial nitric oxide synthase: a paradigm for pterin function involving a novel metal center.

Nitric oxide, a key signaling molecule, is produced by a family of enzymes collectively called nitric oxide synthases (NOS). Here, we report the crystal structure of the heme domain of endothelial NOS in tetrahydrobiopterin (H4B)-free and -bound forms at 1.95 A and 1.9 A resolution, respectively. In both structures a zinc ion is tetrahedrally coordinated to pairs of symmetry-related cysteine residues at the dimer interface. The phylogenetically conserved Cys-(X)4-Cys motif and its strategic location establish a structural role for the metal center in maintaining the integrity of the H4B-binding site. The unexpected recognition of the substrate, L-arginine, at the H4B site indicates that this site is poised to stabilize a positively charged pterin ring and suggests a model involving a cationic pterin radical in the catalytic cycle.

Amino Acid Sequence↗

PubMed1998

Identification of two electron-transfer sites in ascorbate peroxidase using chemical modification, enzyme kinetics, and crystallography.

Chemical and mutagenic modification combined with X-ray crystallography has been used to probe the ascorbate binding site in ascorbate peroxidase (APX). Chemical modification of the single Cys residue in APX with Ellman's reagent (DTNB) blocks the ability of APX to oxidize ascorbate but not other small aromatic phenolic substrates. DTNB-modified APX (APX-TNB) exhibits only 1.3% wild-type activity when ascorbate is used as the substrate but full activity when aromatic substrates, guaiacol or pyrogallol, are used. Stopped-flow studies show that APX-TNB reacts normally with peroxide to give compound I but that the rates of reduction of both compounds I and II by ascorbate are dramatically slowed. Conversion of Cys32 to Ser leads to approximately 70% drop in ascorbate peroxidase activity with no effect on guaiacol peroxidase activity. These results indicate that uncharged aromatic substrates and the anionic ascorbate molecule interact with different sites on APX. The 2.0 A X-ray crystal structure of APX-TNB shows clear electron density for the TNB group covalently attached to Cys32 in all four molecules of the asymmetric unit, indicating complete and specific modification. It appears that the ascorbate site is blocked by DTNB modification which is well removed from the exposed delta-heme edge where aromatic substrates are thought to bind. This is the first experimental evidence indicating that ascorbate oxidation does not occur at the exposed heme edge but at an alternate binding site in the vicinity of Cys32 near Arg172 and the heme propionates.

Ascorbate Peroxidases↗

PubMed1998

Understanding the role of the essential Asp251 in cytochrome p450cam using site-directed mutagenesis, crystallography, and kinetic solvent isotope effect.

Proton transfer in cytochromes P450 is a critical step in the activation of molecular oxygen. Extensive study of the P450cam active site has identified several residues that play a central role in dioxygen bond scission. A highly conserved carboxylate, aspartate-251 in P450cam in the distal helix I, participates in a series of hydrogen-bond/ion pairs near the molecular surface and has been implicated in the catalytic mechanism. Mutation of Asp251 is known to lower activity by 2 orders of magnitude and change the rate-limiting step in the catalytic cycle, suggesting a role for an acid functionality in generation of iron-oxygen reactive intermediates. The turnover rates of the Asp251Asn mutant in various protium-deuterium mixtures have been determined and show a significantly larger kinetic solvent isotope effect, with an overall magnitude of 10 compared to 1.8 for the wild-type P450cam. In addition, a much larger number of protons are involved in the rate-limiting step for the Asp251Asn mutant than in the wild-type enzyme. These results indicate that Asp251 is an essential part of the normal proton delivery machinery required for O-O bond scission. The crystal structure of the Aps251Asn mutant obtained from data collected at cryogenic temperatures has been refined to 1.9 A. Key hydrogen bonds required to hold Asp251 in position have been broken which allows the mutant Asn251 side chain to swing out and away from the O2 binding site leading to a more open active site. This change could allow easier access by water and thus contribute to the observed kinetic solvent isotope effects.

Asparagine↗

PubMed1998

Spectroscopic characterization of recombinant pea cytosolic ascorbate peroxidase: similarities and differences with cytochrome c peroxidase.

Recombinant pea cytosolic ascorbate peroxidase (APX) has been characterized by resonance Raman (RR) and electronic absorption spectroscopies. The ferric and ferrous forms together with the complexes with fluoride and imidazole have been studied and compared with the corresponding spectra of cytochrome c peroxidase (CCP). Ferric APX at neutral pH is a mixture of 6- and 5-coordinate high-spin and 6-c low-spin hemes, the latter two species being dominant. The results suggest that the low-spin form derives from a water/hydroxo ligand bound to the heme iron and not from a strong internal ligand as observed in CCP at alkaline pH. Two Fe-Im stretching modes are identified, as in CCP, but the RR frequencies confirm a weaker His163-Asp208 hydrogen bond than in CCP, as suggested on the basis of the X-ray structure [Patterson, W. R., and Poulos, T. L. (1995) Biochemistry 34, 4331-4341]. The data show that CCP and APX have markedly different orientations of the vinyl substituents on the heme chromophore resulting from different steric constraints exerted by the protein matrix.

Ascorbate Peroxidases↗

PubMed1998

NO news is good news.

The recent determination of the crystal structure of microsomal cytochrome P450 reductase, a diflavin protein that shuttles electrons from NADPH to the P450 heme, represents a significant advance towards the understanding of cytochromes P450. A similar advance was made in a related enzyme system, nitric oxide synthase (NOS). The crystal structure of the NOS heme domain reveals a very different architecture to that observed in P450s and offers significant insight into the production of nitric oxide, one of nature's most important regulatory molecules.

Binding Sites↗

PubMed1998

Crystallization of recombinant human heme oxygenase-1.

Heme oxygenase catalyzes the NADPH, O2, and cytochrome P450 reductase dependent oxidation of heme to biliverdin and carbon monoxide. One of two primary isozymes, HO-1, is anchored to the endoplasmic reticulum membrane via a stretch of hydrophobic residues at the C-terminus. While full-length human HO-1 consists of 288 residues, a truncated version with residues 1-265 has been expressed as a soluble active enzyme in Escherichia coli. The recombinant enzyme crystallized from ammonium sulfate solutions but the crystals were not of sufficient quality for diffraction studies. SDS gel analysis indicated that the protein had undergone proteolytic degradation. An increase in the use of protease inhibitors during purification eliminated proteolysis, but the intact protein did not crystallize. N-terminal sequencing and mass spectral analysis of dissolved crystals indicated that the protein had degraded to two major species consisting of residues 1-226 and 1-237. Expression of the 1-226 and 1-233 versions of human HO-1 provided active enzyme that crystallizes in a form suitable for diffraction studies. These crystals belong to space group P2(1), with unit cell dimensions a = 79.3 A, b = 56.3 A, c = 112.8 A, and beta = 101.5 degrees.

Biliverdine↗

PubMed1998

The role of quaternary interactions on the stability and activity of ascorbate peroxidase.

Point mutations at the dimer interface of the homodimeric enzyme ascorbate peroxidase (APx) were constructed to assess the role of quaternary interactions in the stability and activity of APx. Analysis of the APx crystal structure shows that Glu112 forms a salt bridge with Lys20 and Arg24 of the opposing subunit near the axis of dyad symmetry between the subunits. Two point mutants, E112A and E112K, were made to determine the effects of a neutral (alanine) and repulsive (lysine) mutation on dimerization, stability, and activity. Gel filtration analysis indicated that the ratio of the monomer to dimer increased as the dimer interface interactions went from attractive to repulsive. Differential scanning calorimetry (DSC) data exhibited a decrease in both the transition temperature (Tm) and enthalpy of unfolding (deltaHc) with Tm = 58.3 +/- 0.5 degrees C, 56.0 +/- 0.8 degrees C, and 53.0 +/- 0.9 degrees C and deltaHc = 245 +/- 29 kcal/mol, 199 +/- 38 kcal/mol, and 170 +/- 25 kcal/mol for wild-type APx, E112A, and E112K, respectively. Similar changes were observed based on thermal melting curves obtained by absorption spectroscopy. No change in enzyme activity was found for the E112A mutant, and only a 25% drop in activity was observed for the E112K mutant which demonstrates that the non-Michaelis Menten kinetics of APx is not due to the APx oligomeric structure. The cryogenic crystal structures of the wild-type and mutant proteins show that mutation induced changes are limited to the dimer interface including an alteration in solvent structure.

Ascorbate Peroxidases↗

PubMed1998

Stereochemistry of the chloroperoxidase active site: crystallographic and molecular-modeling studies.

BACKGROUND: Chloroperoxidase (CPO) is the most versatile of the known heme enzymes. It catalyzes chlorination of activated C-H bonds, as well as peroxidase, catalase and cytochrome P450 reactions, including enantioselective epoxidation. CPO contains a proximal heme-thiolate ligand, like P450, and polar distal pocket, like peroxidase. The substrate-binding site is formed by an opening above the heme that enables organic substrates to approach the activated oxoferryl oxygen atom. CPO, unlike other peroxidases, utilizes a glutamate acid-base catalyst, rather than a histidine residue. RESULTS: The crystal structures of CPO complexed with exogenous ligands, carbon monoxide, nitric oxide, cyanide and thiocyanate, have been determined. The distal pocket discriminates ligands on the basis of size and pKa. The refined CPO-ligand structures indicate a rigid active-site architecture with an immobile glutamate acid-base catalyst. Molecular modeling and dynamics simulations of CPO with the substrate cis-beta methylstyrene and the corresponding epoxide products provide a structural and energetic basis for understanding the enantioselectivity of CPO-catalyzed epoxidation reactions. CONCLUSIONS: The various CPO-ligand structures provide the basis for a detailed stereochemical mechanism of the formation of the intermediate compound I, in which Glu183 acts as an acid-base catalyst. The observed rigidity in the active site also explains the relative instability of CPO compound I and the formation of the HOCI chlorinating species. Energetics of CPO-substrate/ product molecular modeling provides a theoretical basis for the P450-type enantioselective epoxidation activities of CPO.

Binding Sites↗

PubMed1997

Redox control of the catalytic cycle of flavocytochrome P-450 BM3.

Flavocytochrome P-450 BM3 from Bacillus megaterium is a 119 kDa polypeptide whose heme and diflavin domains are fused to produce a catalytically self-sufficient fatty acid monooxygenase. Redox potentiometry studies have been performed with intact flavocytochrome P-450 BM3 and with its component heme, diflavin, FAD, and FMN domains. Results indicate that electron flow occurs from the NADPH donor through FAD, then FMN and on to the heme center where fatty acid substrate is bound and monooxygenation occurs. Prevention of futile cycling of electrons is avoided through an increase in redox potential of more than 100 mV caused by binding of fatty acids to the active site of P-450. Redox potentials are little altered for the component domains with respect to their values in the larger constructs, providing further evidence for the discrete domain organization of this flavocytochrome. The reduction potentials of the 4-electron reduced diflavin domain and 2-electron reduced FAD domain are considerably lower than those for the blue FAD semiquinone species observed during reductive titrations of these enzymes and that of the physiological electron donor (NADPH), indicating that the FAD hydroquinone is thermodynamically unfavorable and does not accumulate under turnover conditions. In contrast, the FMN hydroquinone is thermodynamically more favorable than the semiquinone.

Bacillus megaterium↗

PubMed1997

Crystal structures of substrate binding site mutants of manganese peroxidase.

Manganese peroxidase (MnP), an extracellular heme enzyme from the lignin-degrading basidiomycetous fungus, Phanerochaete chrysosporium, catalyzes the oxidation of MnII to MnIII. The latter, acting as a diffusible redox mediator, is capable of oxidizing a variety of lignin model compounds. The proposed MnII binding site of MnP consists of a heme propionate, three acidic ligands (Glu-35, Glu-39, and Asp-179), and two water molecules. Using crystallographic methods, this binding site was probed by altering the amount of MnII bound to the protein. Crystals grown in the absence of MnII, or in the presence of EDTA, exhibited diminished electron density at this site. Crystals grown in excess MnII exhibited increased electron density at the proposed binding site but nowhere else in the protein. This suggests that there is only one major MnII binding site in MnP. Crystal structures of a single mutant (D179N) and a double mutant (E35Q,D179N) at this site were determined. The mutant structures lack a cation at the MnII binding site. The structure of the MnII binding site is altered significantly in both mutants, resulting in increased access to the solvent and substrate.

Basidiomycota↗