Search PubMed⌕ Search

Biomedical subjects

T L Poulos

Publications and source records attributed to T L Poulos.

At least 55 records · Page 3Linked to original sources
PubMed1997

The domain architecture of cytochrome P450BM-3.

Cytochromes P450 utilize redox partners to deliver electrons from NADPH/NADH to the P450 heme center. Microsomal P450s utilize an FAD/FMN reductase. The bacterial fatty acid hydroxylase, P450BM-3, is similar except the P450 heme and FAD/FMN proteins are linked together in a single polypeptide chain arranged as heme-FMN-FAD. Sequence comparisons indicate that the P450BM-3 FMN and FAD domains are similar to flavodoxin and ferredoxin reductase, respectively. Previous work has shown that the heme and FMN/FAD domains can be separately expressed and purified. In this study we have expressed, purified, and characterized the following additional domains: heme-FMN, FMN, and FAD. Each domain retains their prosthetic groups although the FMN domain is more labile. The FAD domain retains a high level of ferricyanide reductase activity but no cytochrome c reductase activity. In addition, we have deleted a 110-residue stretch in the FAD domain that is not present in ferredoxin reductase. This protein retains both FAD and heme but not FMN. We also have investigated the dimerization pattern of the individual domains that lead to the following conclusions. Holo-P450BM-3 appears to dimerize via interactions that do not involve disulfide bond formation, whereas the reductase and FAD domains form intermolecular disulfides. This indicates that the Cys residues not available for dimerization in holo-P450BM-3 are unmasked in the individual domains.

Amino Acid Sequence↗
PubMed1997

Electron transfer between the FMN and heme domains of cytochrome P450BM-3. Effects of substrate and CO.

Cytochrome P450BM-3 has the P450 heme domain and FAD/FMN reductase domain linked together in a single polypeptide chain arranged as heme-FMN-FAD. In the accompanying article (Govindaraj, S., and Poulos, T. L. (1997) J. Biol. Chem. 272, 7915-7921, we have described the preparation and characterization of the various domains of cytochrome P450BM-3. One reason for undertaking this study was to provide simpler systems for studying intramolecular electron transfer reactions. In particular, the heme-FMN version of P450BM-3 that is missing the FAD domain should prove useful in studying the FMN-to-heme electron transfer reaction. This version of P450BM-3 has been designated truncated P450BM-3 or BM3t. In this study we have used laser flash photolysis techniques to generate the reduced semiquinone of 5-deazariboflavin which in turn reduces the FMN of BM3t to the semiquinone, FMN-, at a rate constant of 6600 s-1, whereas the heme is not reduced by the 5-deazariboflavin radical. The reduction of the heme by FMN- does not proceed in the absence of carbon monoxide (CO), whereas in the presence of CO the FMN- to heme electron transfer rate constant is 18 s-1. If a fatty acid substrate is present, this rate constant increases to 250 s-1. Somewhat surprisingly, the rate of heme reduction also is dependent on [CO] which indicates that CO causes some change within the heme pocket and/or interaction between the heme and FMN domains that is required for intramolecular electron transfer.

Bacterial Proteins↗
PubMed1997

Structure of cytochrome P450eryF: substrate, inhibitors, and model compounds bound in the active site.

Much of our understanding of P450 reaction mechanisms derives from studies on P450cam, a bacterial camphor hydroxylase. P450cam has served as the model for understanding detailed structure/function relationships in mammalian P450 enzymes, which have not proved amenable to x-ray crystallographic techniques. To expand and improve the P450 model, we solved the structure of P450eryF, a cytochrome P450 involved in erythromycin biosynthesis. The overall structure of P450eryF is similar to that of P450cam, but differs in the exact positioning of several alpha-helices, which results in the enlargement of the substrate-binding pocket. P450eryF also differs from P450cam in having alanine in place of the highly conserved threonine residue in the active site. To assess the role of this alanine residue, two mutant forms of P450eryF and a substrate analog were examined. Our findings suggest that P450eryF has evolved an active site that utilizes the substrate to assist in catalysis. In addition, the enlarged substrate binding pocket of P450eryF enables P450eryF to bind certain steroid compounds and azole-based steroid hydroxylase inhibitors. Crystals have been obtained for P450eryF complexed with the antifungal drug ketoconazole, and the high-resolution structure has been determined.

Actinomyces↗
PubMed1997

Aspartate residue 7 in amyloid beta-protein is critical for classical complement pathway activation: implications for Alzheimer's disease pathogenesis.

Fibrillar amyloid beta-protein has been implicated in the pathogenesis of Alzheimer's disease because of its neurotoxicity and its ability to activate complement. Reactive microglia, astrocytes and complement (C') components (reviewed in ref. 6) are associated with senile plaques, the fibrillar, beta-sheet assemblies of amyloid beta-peptide found predominantly in brain from individuals with AD (ref. 7). These indications of inflammatory events are not prevalent in the nonfibrillar "diffuse" plaques often seen in age-matched control cases without dementia. Clinical studies over the past several years have correlated the use of anti-inflammatory drugs with a decrease in the incidence and progression of AD dementia and/or dysfunction, supporting a role for gliosis and inflammation in AD pathogenesis (reviewed in ref. 6). C5a, a product of C' activation, is chemotactic for microglia. Thus, complement activation provides a specific mechanism for recruiting reactive glial cells to the site of the fibrillar amyloid beta-protein plaque, which could lead to inflammatory events, neuronal dysfunction and degeneration. With the use of truncated amyloid beta-peptides, the region of amyloid beta-protein limited by residues 4 and 11 has been identified as critical in the interaction between amyloid beta-protein and C1q, the recognition component of the classical complement pathway (CCP), which results in the activation of C'. Furthermore, substitution of an isoaspartic acid for aspartic acid at amyloid beta-protein residue 7 resulted in the complete elimination of CCP-activating activity. A molecular model of this interaction has been generated that should be useful in the design of candidate therapeutic inhibitors of CCP activation by amyloid beta-protein.

Alzheimer Disease↗
PubMed1997

The structure of the cytochrome p450BM-3 haem domain complexed with the fatty acid substrate, palmitoleic acid.

The substrate-bound structures of two cytochrome P450s, P450cam and P450eryF, are known. While these structures reveal important features that control substrate specificity, the problem of how conformational changes allow for substrate entry and product release remains unsolved. The structure of the haem domain of the bacterial fatty acid hydroxylase, P450BM-3, previously was solved in the substrate-free form. Unlike the substrate-bound P450cam and P450eryF structures, the substrate access channel is open in substrate-free P450BM-3. Here we present the X-ray structure of P450BM-3 at 2.7 A bound with a fatty acid substrate, palmitoleic acid. A comparison of the substrate-bound and -free forms reveals major conformational differences and provides the first detailed picture of substrate-induced conformational changes in a P450.

Amino Acid Sequence↗
PubMed1997

Crystal structure of horseradish peroxidase C at 2.15 A resolution.

The crystal structure of horseradish peroxidase isozyme C (HRPC) has been solved to 2.15 A resolution. An important feature unique to the class III peroxidases is a long insertion, 34 residues in HRPC, between helices F and G. This region, which defines part of the substrate access channel, is not present in the core conserved fold typical of peroxidases from classes I and II. Comparison of HRPC and peanut peroxidase (PNP), the only other class III (higher plant) peroxidase for which an X-ray structure has been completed, reveals that the structure in this region is highly variable even within class III. For peroxidases of the HRPC type, characterized by a larger FG insertion (seven residues relative to PNP) and a shorter F' helix, we have identified the key residue involved in direct interactions with aromatic donor molecules. HRPC is unique in having a ring of three peripheral Phe residues, 142, 68 and 179. These guard the entrance to the exposed haem edge. We predict that this aromatic region is important for the ability of HRPC to bind aromatic substrates.

Amino Acid Sequence↗
PubMed1996

An engineered cation site in cytochrome c peroxidase alters the reactivity of the redox active tryptophan.

The crystal structures of cytochrome c peroxidase and ascorbate peroxidase are very similar, including the active site architecture. Both peroxidases have a tryptophan residue, designated the proximal Trp, located directly adjacent to the proximal histidine heme ligand. During the catalytic cycle, the proximal Trp in cytochrome c peroxidase is oxidized to a cation radical. However, in ascorbate peroxidase, the porphyrin is oxidized, not the proximal Trp, despite the close similarity between the two peroxidase active site structures. A cation located approximately 8 A from the proximal Trp in ascorbate peroxidase but absent in cytochrome c peroxidase is thought to be one reason why ascorbate peroxidase does not form a Trp radical. Site-directed mutagenesis has been used to introduce the ascorbate peroxidase cation binding site into cytochrome c peroxidase. Crystal structures show that mutants now bind a cation. Electron paramagnetic resonance spectroscopy shows that the cation-containing mutants of cytochrome c peroxidase no longer form a stable Trp radical. The activity of the cation mutants using ferrocytochrome c as a substrate is < 1% of wild type levels, while the activity toward a small molecule substrate, guaiacol, increases. These results demonstrate that long range electrostatic effects can control the reactivity of a redox active amino acid side chain and that oxidation/reduction of the proximal Trp is important in the oxidation of ferrocytochrome c.

Animals↗
PubMed1996

Probing the cytochrome c peroxidase-cytochrome c electron transfer reaction using site specific cross-linking.

Engineered cysteine residues in yeast cytochrome c peroxidase (CCP) and yeast iso-1-cytochrome c have been used to generate site specifically cross-linked peroxidase-cytochrome c complexes for the purpose of probing interaction domains and the intramolecular electron transfer reaction. Complex 2 was designed earlier [Pappa, H.S., & Poulos, T.L. (1995) Biochemistry 34, 6573-6580] to mimic the known crystal structure of the peroxidase-cytochrome c noncovalent complex [Pelletier, H., & Kraut, J. (1992) Science 258, 1748-1755]. Complex 3 was designed such that cytochrome c is tethered to a region of the peroxidase near Asp148 which has been suggested to be a second site of interaction between the peroxidase and cytochrome c. Using stopped flow methods, the rate at which the ferrocytochrome c covalently attached to the peroxidase transfers an electron to peroxidase compound I is estimated to be approximately 0.5-1 s-1 in complex 3 and approximately 800 s-1 in complex 2. In both complexes the Trp191 radical and not the Fe4+=O oxyferryl center of compound I is reduced. Conversion of Trp191 to Phe slows electron transfer about 10(3) in complex 2. Steady state kinetic measurements show that complex 3 behaves like the wild type enzyme when either horse heart or yeast ferrocytochrome c is used as an exogenous substrate, indicating that the region blocked in complex 3 is not a functionally important interaction site. In contrast, complex 2 is inactive toward horse heart ferrocytochrome c at all ionic strengths tested and yeast ferrocytochrome c at high ionic strengths. Only at low ionic strengths and low concentrations of yeast ferrocytochrome c does complex 2 give wild type enzyme activity. This observation indicates that in complex 2 the primary site of interaction of CCP with horse heart and yeast ferrocytochrome c at high ionic strengths is blocked. The relevance of these results to the pathway versus distance models of electron transfer and to the interaction domains between peroxidase and cytochrome c is discussed.

Animals↗
PubMed1996

The crystal structure of peanut peroxidase.

BACKGROUND: Peroxidases catalyze a wide variety of peroxide-dependent oxidations. Based on sequence alignments, heme peroxidases have been divided into three classes. Crystal structures are available for peroxidases of classes I and II, but until now no structure has been determined for class III, the classical extracellular plant peroxidases. RESULTS: The crystal structure of peanut peroxidase has been solved to 2.7 A resolution. The helical fold is similar to that of known peroxidase structures. The 294-residue polypeptide chain is accompanied by a heme and two calcium ions, and there is some evidence of glycosylation. CONCLUSIONS: This is the first complete structure of a class III peroxidase and as such should serve as a model for other class III enzymes including the much-studied horseradish peroxidase. It may also aid in the interpretation of functional differences between the peroxidase classes. Ten helices conserved in class I and II peroxidases are also found in peanut peroxidase. Key residues of the heme environment and the location of two calcium ions are shared with class II peroxidases. Peanut peroxidase contains three unique helices, two of which contribute to the substrate access channel leading to the heme edge.

Amino Acid Sequence↗
PubMed1996

Probing the structure of the linker connecting the reductase and heme domains of cytochrome P450BM-3 using site-directed mutagenesis.

Cytochrome P450BM-3 is a catalytically self-sufficient fatty acid hydroxylase containing one equivalent each of heme, FMN, and FAD. The heme and flavins reside in separate domains connected by a linker peptide. In an earlier study (Govindaraj S, Poulos T, 1995, Biochemistry 34:11221-11226), we found that the length but not the sequence of the linker connecting the heme and reductase domains is important for enzyme activity. In the present study, residues in the linker were replaced with Pro and Gly to probe the role that regular secondary structure plays in linker function. The rate of flavin-to-heme electron transfer and the fatty acid hydroxylase activities of the glycine and proline substitution mutants, including a six-proline substitution, did not change significantly relative to wild-type enzyme. These results indicate that the linker does not adopt any regular secondary structure essential for activity and that the length of the linker is the critical feature that controls flavin-to-heme electron transfer.

Bacterial Proteins↗
PubMed1996

Conformational dynamics in cytochrome P450-substrate interactions.

There now are four known cytochrome P450 crystal structures. Two of these, P450cam and P450eryF, are substrate-bound while P450terp and the heme domain of P450BM-3 are substrate-free. Here we describe a preliminary analysis of the P450BM-3 heme domain complexed with the 16-carbon fatty acid substrate, palmitoleic acid. A comparison of the substrate-free and -bound structures shows that a large conformational change in the substrate access channel accompanies substrate binding. This new information, together with the substrate-bound structures of P450cam and P450eryF, reveals which regions of P450 are the most important in controlling the dynamics of substrate binding and recognition.

Bacterial Proteins↗
PubMed1996

Substrate-assisted catalysis in cytochrome P450eryF.

A highly conserved threonine in the active site of cytochromes P450 has been proposed to participate in O2 binding and cleavage. Cytochrome P450eryF is unusual in having alanine in place of this threonine and an ordered active site water molecule (Wat 519) which is hydrogen bonded to the substrate 5-hydroxyl group and is in position to operate as an acid catalyst required for cleaving dioxygen. To asses the role of this alanine residue and Wat 519 in catalysis, two mutant forms of P450eryF (Ala --> Ser,Ala --> Thr) and a substrate analogue lacking a 5-hydroxyl group were examined using kinetic, spectral and crystallographic techniques. In each case decreased catalytic activity was correlated with a loss or repositioning of Wat 519. These findings suggest that P450eryF utilizes the substrate to assist in the acid-catalysed dioxygen bond cleavage reaction.

Bacterial Proteins↗
PubMed1995

The crystal structure of chloroperoxidase: a heme peroxidase--cytochrome P450 functional hybrid.

BACKGROUND: Chloroperoxidase (CPO) is a versatile heme-containing enzyme that exhibits peroxidase, catalase and cytochrome P450-like activities in addition to catalyzing halogenation reactions. The structure determination of CPO was undertaken to help elucidate those structural features that enable the enzyme to exhibit these multiple activities. RESULTS: Despite functional similarities with other heme enzymes, CPO folds into a novel tertiary structure dominated by eight helical segments. The catalytic base, required to cleave the peroxide O-O bond, is glutamic acid rather than histidine as in other peroxidases. CPO contains a hydrophobic patch above the heme that could be the binding site for substrates that undergo P450-like reactions. The crystal structure also shows extensive glycosylation with both N- and O-linked glycosyl chains. CONCLUSIONS: The proximal side of the heme in CPO resembles cytochrome P450 because a cysteine residue serves as an axial heme ligand, whereas the distal side of the heme is 'peroxidase-like' in that polar residues form the peroxide-binding site. Access to the heme pocket is restricted to the distal face such that small organic substrates can interact with the iron-linked oxygen atom which accounts for the P450-like reactions catalyzed by chloroperoxidase.

Amino Acid Sequence↗
PubMed1995

Proton NMR studies of cytochrome c peroxidase mutant N82A: hyperfine resonance assignments, identification of two interconverting enzyme ofecies, quantitating the rate of interconversion, and determination of equilibrium constants.

The cyanide-ligated form of the baker's yeast cytochrome c peroxidase mutant bearing the mutation Asn82-->Ala82 ([N82A]CcPCN) has been studied by proton NMR spectroscopy. This mutation alters an amino acid that forms a hydrogen bond to His52, the distal histidine residue that interacts in the heme pocket with heme-bound ligands. His52 is a residue critical to cytochrome c peroxidase's normal function. Proton hyperfine resonance assignments have been made for the cyanide-ligated form of the mutant by comparison with 1-D and NOESY spectra of the wild-type native enzyme. For [N82A]CcPCN, proton NMR spectra reveal two significant phenomena. First, similar to results published for the related mutant [N82D]CcPCN [Satterlee, J. D., et al. (1994) Eur. J. Biochem. 244, 81-87], for Ala82 mutation disrupts the hydrogen bond between His52 and the heme-ligated CN. Second, four of the 24 resolved hyperfine-shifted resonances are doubled in the mutant enzyme's proton spectrum, leading to the concept that the heme active site environment is dynamically microheterogeneous on a very localized scale. Two magnetically inequivalent enzyme forms are detected in a pure enzyme preparation. Varying temperature causes the two enzyme forms to interconvert. Magnetization transfer experiments further document this interconversion between enzyme forms and have been used to determine that the rate of interconversion is 250 (+/- 53) s-1. The equilibrium constant at 20 degrees C is 1.5. Equilibrium constants have been calculated at various temperatures between 5 and 29 degrees C leading to the following values: delta H = 60 kJ mol-1; delta S = 0.20 kJ K-1 mol-1.

Binding Sites↗
PubMed1995

The role of Thr268 in oxygen activation of cytochrome P450BM-3.

Cytochrome P450BM-3, a catalytically self-sufficient monooxygenase from Bacillus megaterium, catalyzes the omega-n (n = 1-3) hydroxylation of fatty acids in the presence of O2 and NADPH. Like most other P450s, cytochrome P450BM-3 contains a threonine residue (Thr268) in the distal I helix thought to be important for O2 binding and activation. Thr268 has been converted to alanine and the enzymatic properties and heme domain crystal structure determined. Using sodium laurate as the substrate, the mutant exhibited slower rates of O2 and NADPH consumption. In addition, electron transfer is uncoupled from substrate hydroxylation as evidenced by the greater production of water and peroxide in the mutant compared to the wild-type enzyme. The crystal structure of the mutant reveals that the only changes in structure are confined to the site of mutation. These data indicate an important role for Thr268 in O2 binding and activation in the metabolism of sodium laurate by cytochrome P450BM-3.

Bacillus megaterium↗
PubMed1995

Effect of the His175-->Glu mutation on the heme pocket architecture of cytochrome c peroxidase.

Resonance Raman (RR) and electronic absorption spectra of the ferric and ferrous forms of the His175Glu mutant of cytochrome c peroxidase are reported. At 296 K, the FeIII form is five-coordinate high spin and the resonance Reman spectra are very similar to those obtained for the wild type enzyme, even though in the mutant the Fe atom is bound to an oxygen atom of the Glu residue. The only difference is that the bands due to the out-of-plane modes are very weak, indicating a less distorted heme plane compared to CCP. The absorption spectrum is similar to that of CCP, as far as the Soret and alpha, beta bands are concerned, but the charge-transfer band due to the a2u(pi)-->eg(d pi) transition is 8 nm blue-shifted relative to that of the wild type enzyme, indicating that a more negative ligand is bound to the heme iron. As the temperature is lowered, the five-coordinate heme converts to a six-coordinate high-spin form. The conversion is readily reversible. A temperature effect on the protein structure is proposed that permits the Fe atom to approach the heme plane and to bind the distal water molecule. The results are discussed in terms of the X-ray structure, which shows a different disposition of the distal water molecules in the Glu175 mutant. The RR spectra also show that the heme is more contracted and distorted at 19 K than at room temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗