共查询到20条相似文献,搜索用时 15 毫秒
1.
Mason MG Nicholls P Divne C Hallberg BM Henriksson G Wilson MT 《Biochimica et biophysica acta》2003,1604(1):47-54
Phanerochaete chrysosporium cellobiose oxidoreductase (CBOR) comprises two redox domains, one containing flavin adenine dinucleotide (FAD) and the other protoheme. It reduces both two-electron acceptors, including molecular oxygen, and one-electron acceptors, including transition metal complexes and cytochrome c. If the latter reacts with the flavin, the reduced heme b acts merely as a redox buffer, but if with the b heme, enzyme action involves a true electron transfer chain. Intact CBOR fully reduced with cellobiose, CBOR partially reduced by ascorbate, and isolated ascorbate-reduced heme domain, all transfer electrons at similar rates to cytochrome c. Reduction of cationic one-electron acceptors via the heme group supports an electron transfer chain model. Analogous reactions with natural one-electron acceptors can promote Fenton chemistry, which may explain evolutionary retention of the heme domain and the enzyme's unique character among secreted sugar dehydrogenases. 相似文献
2.
The active site of cellobiose dehydrogenase from Phanerochaete chrysosporium is composed of two subsites, a catalytic C subsite and a substrate-binding B subsite. Based on the crystal structure of the enzyme with a cellobiose analogue, residue Glu279 was selected for site-directed mutagenesis studies. Substitution of Glu279 to Ala, Asn, and Asp had no effect on the expression of the protein in Pichia pastoris but completely abolished its enzymatic activity. Substitution of Glu279 to Gln drastically altered the enzyme’s substrate specificity. While the wild-type cellobiose dehydrogenase efficiently oxidizes cellobiose and lactose, the Glu279Gln mutant retained most of its activity with cellobiose but was completely inactive with lactose. We generated structural models of the active site interacting with cellobiose and lactose to provide an interpretation of these results. 相似文献
3.
Synthesis and bacterial expression of a gene encoding the heme domain of assimilatory nitrate reductase 总被引:1,自引:0,他引:1
Barber MJ Desai SK Marohnic CC Hernandez HH Pollock VV 《Archives of biochemistry and biophysics》2002,402(1):38-50
Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex Mo-pterin-, cytochrome b(557)-, and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. A codon-optimized gene has been synthesized for expression of the central cytochrome b(557)-containing fragment, corresponding to residues A542-E658, of spinach assimilatory nitrate reductase. While expression of the full-length synthetic gene in Escherichia coli did not result in significant heme domain production, expression of a Y647* truncated form resulted in substantial heme domain production as evidenced by the generation of "pink" cells. The histidine-tagged heme domain was purified to homogeneity using a combination of NTA-agarose and size-exclusion FPLC, resulting in a single protein band following SDS-PAGE analysis with a molecular mass of approximately 13 kDa. MALDI-TOF mass spectrometry yielded an m/z ratio of 12,435 and confirmed the presence of the heme prosthetic group (m/z=622) while cofactor analysis indicated a 1:1 heme to protein stoichiometry. The oxidized heme domain exhibited spectroscopic properties typical of a b-type cytochrome with a visible Soret maximum at 413 nm together with epr g-values of 2.98, 2.26, and 1.49, consistent with low-spin bis-histidyl coordination. Oxidation-reduction titrations of the heme domain indicated a standard midpoint potential (E(o)') of -118 mV. The isolated heme domain formed a 1:1 complex with cytochrome c with a K(A) of 7 microM (micro=0.007) and reconstituted NADH:cytochrome c reductase activity in the presence of a recombinant form of the spinach nitrate reductase flavin domain, yielding a k(cat) of 1.4 s(-1) and a K(m app) for cytochrome c of 9 microM. These results indicate the efficient expression of a recombinant form of the heme domain of spinach nitrate reductase that retained the spectroscopic and thermodynamic properties characteristic of the corresponding domain in the native spinach enzyme. 相似文献
4.
An analysis of substrate binding interactions in the heme peroxidase enzymes: A structural perspective 总被引:1,自引:0,他引:1
Andrea Gumiero Clive L. Metcalfe Emma Lloyd Raven 《Archives of biochemistry and biophysics》2010,500(1):13-14484
The interactions of heme peroxidase enzymes with their substrates have been studied for many years, but only in the last decade or so has structural information begun to appear. This review looks at crystal structures for a number of heme peroxidases in complex with a number of (mainly organic) substrates. It examines the nature and location of the binding interaction, and explores functional similarities and differences across the family. 相似文献
5.
Electron transfers in photosynthesis and respiration commonly occur between metal-containing cofactors that are separated by large molecular distances. Understanding the underlying physics and chemistry of these biological electron transfer processes is the goal of much of the work in our laboratories. Employing laser flash-quench triggering methods, we have shown that 20 Å, coupling-limited Fe(II) to Ru(III) and Cu(I) to Ru(III) electron tunneling in Ru-modified cytochromes and blue copper proteins can occur on the microsecond timescale both in solutions and crystals; and, further, that analysis of these rates suggests that distant donor-acceptor electronic couplings are mediated by a combination of sigma and hydrogen bonds in folded polypeptide structures. Redox equivalents can be transferred even longer distances by multistep tunneling, often called hopping, through intervening amino acid side chains. In recent work, we have found that 20 Å hole hopping through an intervening tryptophan is several hundred-fold faster than single-step electron tunneling in a Re-modified blue copper protein. 相似文献
6.
The 1-methyl-nicotinamide radical (MNA(*)), produced by pulse radiolysis has previously been shown to reduce the Cu(A)-site of cytochromes aa(3), a process followed by intramolecular electron transfer (ET) to the heme a but not to the heme a(3) [Farver, O., Grell, E., Ludwig, B., Michel, H. and Pecht, I. (2006) Rates and equilibrium of CuA to heme a electron transfer in Paracoccus denitrificans cytochrome c oxidase. Biophys. J. 90, 2131-2137]. Investigating this process in the cytochrome ba(3) of Thermus thermophilus (Tt), we now show that MNA(*) also reduces Cu(A) with a subsequent ET to the heme b and then to heme a(3), with first-order rate constants 11200 s(-1), and 770 s(-1), respectively. The results provide clear evidence for ET among the three spectroscopically distinguishable centers and indicate that the binuclear a(3)-Cu(B) center can be reduced in molecules containing a single reduction equivalent. 相似文献
7.
A promoted electron transfer of an antitumor drug, mitoxantrone (MTX), intercalating into DNA duplex was successfully obtained upon addition of cytochromes c (cyt. c) in NaAc-HAc buffer solution (pH 4.5). The experimental results suggested that co-existence of MTX and cyt. c in the DNA helix is an important factor for accelerated electron transfer of MTX, where the promoter, cyt. c, operated smoothly through the DNA bridge. The UV/Vis spectroscopic experiments further confirmed the interaction process. Furthermore, a possible mechanism of such reaction was also discussed in this paper. 相似文献
8.
Warrilow AG Lamb DC Kelly DE Kelly SL 《Biochemical and biophysical research communications》2002,299(2):189-195
The recently completed genome of the basidiomycete, Phanerochaete chrysosporium, revealed the presence of one NADPH-cytochrome P450 oxidoreductase (CPR; EC 1.6.2.4) gene and >123 cytochrome P450 (CYP) genes. How a single CPR can drive many CYPs is an important area of study. We have investigated this CPR to gain insight into the mechanistic and structural biodiversity of the cytochrome P450 catalytic system. Native CPR and a NH(2)-terminally truncated derivative lacking 23 amino acids have been overexpressed in Escherichia coli and purified to electrophoretic homogeneity. Steady-state kinetics of cytochrome c reductase activity revealed a random sequential bireactant kinetic mechanism in which both products form dead-end complexes reflecting differences in CPR kinetic mechanisms even within a single kingdom of life. Removal of the N-terminal anchor of P. chrysosporium CPR did not alter the kinetic properties displayed by the enzyme in vitro, indicating it was a useful modification for structural studies. 相似文献
9.
Complex III in the mitochondrial electron transport chain is a proposed site for the enhanced production of reactive oxygen species that contribute to aging in the heart. We describe a defect in the ubiquinol binding site (Q(O)) within cytochrome b in complex III only in the interfibrillar population of cardiac mitochondria during aging. The defect is manifested as a leak of electrons through myxothiazol blockade to reduce cytochrome b and is observed whether cytochrome b in complex III is reduced from the forward or the reverse direction. The aging defect increases the production of reactive oxygen species from the Q(O) site of complex III in interfibrillar mitochondria. A greater leak of electrons from complex III during the oxidation of ubiquinol is a likely mechanism for the enhanced oxidant production from mitochondria that contributes to aging in the rat heart. 相似文献
10.
In several classes of proteins the redox center provides an additional intrinsic biophysical probe that could be used to study the protein structure and function. In present report reorganization energy (lambda, as a parameter describing electron transfer properties) was used to study the protein structural changes around the heme prosthetic group in cytochrome c (cyt c). We attempted to monitor the value of this parameter upon the unfolding process of cyt c by urea, during which it was increased sigmoidally from about 0.52 to 0.82 eV for native and unfold protein, respectively. Results indicate that by structural changes in the heme site, lambda provides a complementary tool for following the unfolding process. Assuming a reversible two-state model for cyt c unfolding, Delta G(H2O), Cm and m values were determined to be 8.32+/-0.7 kcal mol(-1), 1.53+/-0.19 kcalmol(-1)M(-1) and 5.03 M, respectively. 相似文献
11.
Respiratory chain complex II has been extensively studied but little is known about its assembly and the role of its heme group. Mutations in the phylogenetically conserved histidine 127 of the SDHC subunit have been shown to abrogate heme binding in yeast and bacteria without impairing complex II assembly or enzymatic activities. Here we show that in mammalian cells these mutations lead to a complete reduction of SDHC in mitochondria, a destabilisation of SDHD and SDHB, and to an abrogation of complex II enzymatic activities, suggesting that in mammalian cells complex II assembly is more complex than in lower organisms. 相似文献
12.
Satoh T Itoga A Isogai Y Kurihara M Yamada S Natori M Suzuki N Suruga K Kawachi R Arahira M Nishio T Fukazawa C Oku T 《FEBS letters》2002,531(3):543-547
To investigate the role of the heme axial ligand in the conformational stability of c-type cytochrome, we constructed M58C and M58H mutants of the red alga Porphyra yezoensis cytochrome c(6) in which the sixth heme iron ligand (Met58) was replaced with Cys and His residues, respectively. The Gibbs free energy change for unfolding of the M58H mutant in water (DeltaG degrees (unf)=1.48 kcal/mol) was lower than that of the wild-type (2.43 kcal/mol), possibly due to the steric effects of the mutation on the apoprotein structure. On the other hand, the M58C mutant exhibited a DeltaG degrees (unf) of 5.45 kcal/mol, a significant increase by 3.02 kcal/mol compared with that of wild-type. This increase was possibly responsible for the sixth heme axial bond of M58C mutant being more stable than that of wild-type according to the heme-bound denaturation curve. Based on these observations, we propose that the sixth heme axial ligand is an important key to determine the conformational stability of c-type cytochromes, and the sixth Cys heme ligand will give stabilizing effects. 相似文献
13.
Khajamohiddin Syed Venkataramanan Subramanian 《Biochemical and biophysical research communications》2010,399(4):492-497
Fungi, particularly the white rot basidiomycetes, have an extraordinary capability to degrade and/or mineralize (to CO2) the recalcitrant fused-ring high molecular weight (?4 aromatic-rings) polycyclic aromatic hydrocarbons (HMW PAHs). Despite over 30 years of research demonstrating involvement of P450 monooxygenation reactions in fungal metabolism of HMW PAHs, specific P450 monooxygenases responsible for oxidation of these compounds are not yet known. Here we report the first comprehensive identification and functional characterization of P450 monooxygenases capable of oxidizing different ring-size PAHs in the model white rot fungus Phanerochaete chrysosporium using a successful genome-to-function strategy. In a genome-wide P450 microarray screen, we identified six PAH-responsive P450 genes (Pc-pah1-Pc-pah6) inducible by PAHs of varying ring size, namely naphthalene, phenanthrene, pyrene, and benzo(a)pyrene (BaP). Using a co-expression strategy, cDNAs of the six Pc-Pah P450s were cloned and expressed in Pichia pastoris in conjunction with the homologous P450 oxidoreductase (Pc-POR). Each of the six recombinant P450 monooxygenases showed PAH-oxidizing activity albeit with varying substrate specificity towards PAHs (3-5 rings). All six P450s oxidized pyrene (4-ring) into two monohydroxylated products. Pc-Pah1 and Pc-Pah3 oxidized BaP (5-ring) to 3-hydroxyBaP whereas Pc-Pah4 and Pc-Pah6 oxidized phenanthrene (3-ring) to 3-, 4-, and 9-phenanthrol. These PAH-oxidizing P450s (493-547 aa) are structurally diverse and novel considering their low overall homology (12-23%) to mammalian counterparts. To our knowledge, this is the first report on specific fungal P450 monooxygenases with catalytic activity toward environmentally persistent and highly toxic HMW PAHs. 相似文献
14.
The operation of the alternative electron-leak pathways mediated by cytochrome c in mitochondria 总被引:3,自引:0,他引:3
Low (1 x 10(-9)M) concentrations of cytochrome c inhibit H2O2 production in cytochrome c-depleted mitochondria, purified succinate-cytochrome c reductase (SCR) and antimycin A inhibited cytochrome c-depleted HMP. At higher concentration (2 x 10(-6)M), cytochrome c eliminates pre-existed H2O2 if feeding electrons to it by succinate. Cytochrome c also decreases the OH* produced by succinate-cytochrome c reductase oxidizing succinate. We conclude that the alternative electron-leak pathway mediated by cytochrome c operates very well. In the presence of antimycin A, ferrocytochrome c can suppress the generation of H2O2 in SCR system, but ferricytochrome c cannot. Similar results are obtained on the elimination of pre-existed H2O2 by cytochrome c. For hydroxyl radical, antimycin A abolishes the suppression caused by both ferrocytochrome c and ferricytochrome c. These results indicate that the reductive state of cytochrome c caused by electron-flow is necessary and sufficient for the operation of cytochrome c-mediated electron-leakage pathway. 相似文献
15.
16.
Alan D. Goddard Julie M. Stevens Feng Rao Despoina A. I. Mavridou Weelee Chan David J. Richardson James W. A. Allen Stuart J. Ferguson 《The Journal of biological chemistry》2010,285(30):22882-22889
The Ccm cytochrome c maturation System I catalyzes covalent attachment of heme to apocytochromes c in many bacterial species and some mitochondria. A covalent, but transient, bond between heme and a conserved histidine in CcmE along with an interaction between CcmH and the apocytochrome have been previously indicated as core aspects of the Ccm system. Here, we show that in the Ccm system from Desulfovibrio desulfuricans, no CcmH is required, and the holo-CcmE covalent bond occurs via a cysteine residue. These observations call for reconsideration of the accepted models of System I-mediated c-type cytochrome biogenesis. 相似文献
17.
The cytochrome c maturation system of Escherichia coli contains two monotopic membrane proteins with periplasmic, functional domains, the heme chaperone CcmE and the thioredoxin CcmG. We show in a domain swap experiment that the membrane anchors of these proteins can be exchanged without drastic loss of function in cytochrome c maturation. By contrast, the soluble periplasmic forms produced with a cleavable OmpA signal sequence have low biological activity. Both the chimerical CcmE (CcmG'-'E) and the soluble periplasmic CcmE produce low levels of holo-CcmE and thus are impaired in their heme receiving capacity. Also, both forms of CcmE can be co-precipitated with CcmC, thus restricting the site of interaction of CcmE with CcmC to the C-terminal periplasmic domain. However, the low level of holo-CcmE formed in the chimera is transferred efficiently to cytochrome c, indicating that heme delivery from CcmE does not involve the membrane anchor. 相似文献
18.
Suruga K Murakami K Taniyama Y Hama T Chida H Satoh T Yamada S Hakamata W Kawachi R Isogai Y Nishio T Oku T 《Biochemical and biophysical research communications》2004,315(4):815-822
To investigate the nitrite reducing activity of microperoxidases (mps) in the presence of methyl viologen and dithionite, the fragments C14-K22 (mp9), V11-L32 (mp22), and G1-M65 (mp65) containing heme were prepared by enzymatic hydrolysis of commercially equine heart cytochrome c (Cyt c), in which His is axially coordinated to heme iron, and acts as its fifth ligand. The nitrite reducing activity of mps was measured under anaerobic condition, and the nitrite reducing activity of mps increased with the cutting of the peptide chain. The activity of the shortest nonapeptide mp9 was approximately 120-fold that of Cyt c (104 amino acid residues) and 3.2-fold that of nitrite reductase (EC 1.7.7.1) from Escherichia coli. In the nitrite reduction by mp, nitrite was completely reduced to ammonia. We presumed that ferrous mps reduced NO2- to NO by donating one electron, the NO was completely reduced to NH4+ under anaerobic condition via ferrous-NO complexes as a reaction intermediate using visible spectra and ESR spectra, and this overall reaction was a 6-electron and 8-proton reduction. Sepharose-immobilized mp9 had a nitrite reducing activity similar to that of mp9 in solution, and the resin retained the activity after five uses and even 1-year storage. The mp will be able to use as a substitute for nitrite reductase. 相似文献
19.
Bacillus subtilis heme A synthase is a membrane protein with 8 transmembrane segments. By using a two-step mutagenesis approach we have generated and selected a fully functional enzyme protein variant with a seven residue internal deletion. The biochemical properties of the shortened variant are similar to those of the normal enzyme. This could indicate that residue H209 in the mutant protein substitutes for the missing H216 as an axial ligand to the heme iron. Our results provide insight in routes of membrane protein evolution and the structure of heme A synthases. 相似文献
20.
The largest part of the bio-decolorization investigations have been performed to date on a single dye without exploring the behavior in complex mixtures as the real dyeing baths. Therefore, mixtures of dyes belonging to azo and anthraquinonic classes, chosen among the most utilized in textile wool dyeing, were employed for comparative enzymatic decolorization studies using the extracellular extracts from the white rot fungus Funalia trogii, to understand how the concomitant presence of more than one dye could influence their degradation course and yield.Fungal extracts containing laccase activity only were capable to partially decolorize dyes mixtures from the different classes analyzed. The deconvolution of the decolorization with time allowed to monitor the degradation of the single dyes in the mixtures evidencing a time dependent differential decolorization not observed for the singles alone. Some dyes in the blend were in fact decolorized only when the most easily converted dyes were largely transformed. These experiments would allow to help the dyeing factories in the selection of the most readily degraded dyes.Since F. trogii grown on different media and activators shows diverse levels of expression of the redox enzymes laccase and cellobiose dehydrogenase (CDH), the dyes mixtures recalcitrant to decolorization by laccase activity alone, were subjected to the combined action of extracts containing laccase and CDH. The use of CDH, in support to the activity of laccase, resulted in substantial decolorization increases (>84%) for all the refractory dyes mixtures. 相似文献