Isolation and characterization of nitric oxide reductase from Paracoccus halodenitrificans

Nitric oxide reductase was isolated from the membrane fraction of a denitrifying bacterium, Paracoccus halodenitrificans, in the presence of n-dodecyl beta-D-maltoside. A relatively simple and effective procedure to purify NO reductase using DEAE-Toyopearl and hydroxyapatite (ceramic) chromatographies has been developed. The enzyme consisted of two subunits with molecular masses of 20 and 42 kDa associated with the c-type heme and two b-type hemes, respectively. The optical and magnetic circular dichroism (MCD) spectra of the oxidized (as isolated) and reduced enzymes indicated that the heme c is in the low-spin state and the hemes b are in the high- and low-spin states. The EPR spectrum also showed the presence of the split high-spin component (g perpendicular = 6.6, 6.0) and two low spin components (gz,y,x = 2.96, 2.26, 1.46, gz = 3.59). Although the presence of an extra iron was suggested from atomic absorption spectroscopy, a non-heme iron could not be detected by colorimetric titrations using ferene and 2-(5-nitro-2-pyridylazo)- 5-(N-propyl-N-sulfopropylamino)phenolate (PAPS). One of the extra signals at g = 4.3 and 2.00 might come from a non-heme iron, while they may originate from an adventitious iron and a certain nonmetallic radical, respectively. When CO acted on the reduced enzyme, both of the low-spin hemes were not affected, and when NO acted on the reduced enzyme, the optical and MCD spectra were of a mixture of the oxidized and reduced enzymes. Consequently, the reduction of NO was supposed to take place at the high-spin heme b. The heme c and the low-spin heme b centers were considered to function as electron mediators during the intermolecular and intramolecular processes.     

Effect of ligand conformation on melanoma cell alpha3beta1 integrin-mediated signal transduction events: implications for a collagen structural modulation mechanism of tumor cell invasion

The importance of three-dimensional interactions between receptors with their respective ligands has been extensively explored during the binding process, but considerably less so for postbinding events such as induction of signaling pathways. Tumor cell receptor association with basement membrane proteins is believed to facilitate the metastatic process. Melanoma and ovarian carcinoma cells have been shown to utilize the alpha3beta1 integrin to bind to models of the alpha1(IV)531-543 sequence from basement membrane (type IV) collagen [Miles, A. J., et al. (1994) J. Biol. Chem. 269, 30939-30945; Miles, A. J., et al. (1995) J. Biol. Chem. 270, 29047-29050]. In the present study, the effects of ligand three-dimensional structure on possible signal transduction pathways induced by alpha3beta1 integrin binding have been evaluated. Human melanoma cell binding to type IV collagen resulted in Tyr phosphorylation of p125(FAK), consistent with prior studies correlating beta1 integrin subunit binding to collagen and p125(FAK) Tyr phosphorylation. Cross-linking of an anti-alpha3 integrin subunit monoclonal antibody also induced p125(FAK) Tyr phosphorylation. Incubation of melanoma cells with single-stranded or triple-helical peptide models of alpha1(IV)531-543 induced Tyr phosphorylation of intracellular proteins. Immunoprecipitation analysis identified one of these proteins as pp125(FAK). Induction of p125(FAK) Tyr phosphorylation was enhanced and the time of induction was shortened when the ligand was used in triple-helical conformation. Subsequent clustering of either the single-stranded or the triple-helical ligand also increased the level of p125(FAK) phosphorylation compared to unclustered ligand. The clustered triple-helical peptide ligand induced more rapid paxillin Tyr phosphorylation than the single-stranded ligand. In addition, the induction of activated proteases was found to be more rapid due to ligand triple helicity. Overall, these studies have shown that (i) a model of an isolated sequence from type IV collagen, alpha1(IV)531-543, can induce alpha3beta1 integrin-mediated signal transduction in melanoma cells and (ii) ligand conformation (secondary, tertiary, and/or quaternary structure) can directly influence several alpha3beta1 integrin-mediated signal transduction events. The effects of ligand conformation suggest that a "collagen structural modulation" mechanism may exist for tumor cell invasion, whereby triple-helical collagen promotes cell binding and induction of signal transduction, subsequently leading to collagen dissolution by proteases, decreased signal transduction, and enhanced tumor cell motility.     

Characterization of C415 mutants of neuronal nitric oxide synthase

Nitric oxide synthase (NOS) catalyzes the oxidation of L-arginine to citrulline and nitric oxide. C415H and C415A mutants of the neuronal isoform of NOS (nNOS) were expressed in a baculovirus system and purified to homogeneity for spectral analysis and activity measurements. UV-visible spectra of each mutant lacked an observable Soret peak, suggesting that neither mutant contained heme. When reduced in the presence of CO, however, a small Soret centered at 417 nm could be detected for the C415H mutant, further supporting the assignment of C415 as the axial ligand to the heme. In addition to a deficiency in bound heme, neither mutant had any detectable bound tetrahydrobiopterin, as compared to wild-type enzyme, which had a ratio of 0.84 mol of bound pteridine:1 mol of nNOS 160 kDa subunit. The C415H mutant contained bound FAD and FMN at levels of 1.0 +/- 0.1 and 0.9 +/- 0.1 mol/mol of nNOS subunit, respectively. UV-visible spectra of both nNOS mutants retained the distinctive absorbance due to tightly associated oxidized flavin prosthetic groups. Further, the spectra suggested the presence of a neutral flavin semiquinone. Ferricyanide oxidation of the C415A mutant yielded a spectrum that was essentially that of oxidized flavin. Ferricyanide titration showed that the C415A mutant contained approximately 1 reducing equiv. Circular dichroism spectra suggested that each mutant was folded properly, in that both spectra were found to be essentially identical to the spectrum of wild-type nNOS. Neither mutant could synthesize nitric oxide, and neither mutant had the ability to oxidize NADPH unless an exogenous electron acceptor was added. The rate of cytochrome c reduction by each mutant was found to be slightly less, but very similar to the rate (approximately 20 mumol mg-1 min-1) observed with wild-type nNOS. In all cases, the rate of cytochrome c reduction increased approximately 15-fold with the addition of calmodulin. Overall, these spectral and activity data suggest that C415 is the axial heme ligand and that a point mutation at C415 prevents binding of heme and tetrahydrobiopterin without interfering with the global folding or the reductase function of nNOS.     

Structural and kinetic studies of the Y73E mutant of octaheme cytochrome c3 (Mr = 26 000) from Desulfovibrio desulfuricans Norway

A combination of structural, kinetic, and interaction experiments has been used to study the role of a highly conserved aromatic residue, Tyr73, parallel to the sixth heme axial ligand of heme 4 in multiheme cytochrome c3 (Mr = 26 000), also called cytochrome cc3 or octaheme cytochrome, from Desulfovibrio desulfuricans Norway. This residue is expected to be involved in intermolecular electron transfer and protein-protein interaction, since heme 4 is described to be the interaction site between physiological partners. The kinetic experiments show that the Y73E replacement provokes no significant change in the electron-transfer reaction with the physiological partner, the [NiFeSe] hydrogenase, but that the protein-protein interaction between cytochrome c3 (Mr = 26 000) and hydrogenase is strongly affected by the mutation. The aromatic residue does not play a role in maintaining the axial heme ligand in a particular orientation, since the mutation did not affect the orientation of histidine 77, the sixth axial ligand of heme 4. The structural analysis by X-ray crystallography clearly shows that a rearrangement of the charged residues in the vicinity of the mutation site is responsible for the change in protein-protein interaction, which is of an electrostatic nature. Lys22 and Arg66, residues which are located at the interacting surface, are twisted toward the mutated position Glu73 in order to compensate for the negative charge and therefore are no longer accessible for the docking with a physiological partner. Tyr73 has instead a structural function and probably a role in maintaining the hydrophobic environment of the heme 4 cavity rather than a function in the intermolecular electron transfer with the physiological partners.     

N5-(1-Imino-3-butenyl)-L-ornithine. A neuronal isoform selective mechanism-based inactivator of nitric oxide synthase

Nitric oxide synthase (NOS) catalyzes the NADPH- and O2-dependent conversion of L-arginine to nitric oxide (NO) and citrulline; three isoforms, the neuronal (nNOS), endothelial, and inducible, have been identified. Because overproduction of NO is known to contribute to several pathophysiological conditions, NOS inhibitors are of interest as potential therapeutic agents. Inhibitors that are potent, mechanism-based, and relatively selective for the NOS isoform causing pathology are of particular interest. In the present studies we report that vinyl-L-NIO (N5-(1-imino-3-butenyl)-L-ornithine; L-VNIO) binds to and inhibits nNOS in competition with L-arginine (Ki = 100 nM); binding is accompanied by a type I optical difference spectrum consistent with binding near the heme cofactor without interaction as a sixth axial heme ligand. Such binding is fully reversible. However, in the presence of NADPH and O2, L-VNIO irreversibly inactivates nNOS (kinact = 0.078 min-1; KI = 90 nM); inactivation is Ca2+/calmodulin-dependent. The cytochrome c reduction activity of the enzyme is not affected by such treatment, but the L-arginine-independent NADPH oxidase activity of nNOS is lost in parallel with the overall activity. Spectral analyses establish that the nNOS heme cofactor is lost or modified by L-VNIO-mediated mechanism-based inactivation of the enzyme. The inducible isoform of NOS is not inactivated by L-VNIO, and the endothelial isoform requires 20-fold higher concentrations to attain approximately 75% of the rate of inactivation seen with nNOS. Among the NOS inactivating L-arginine derivatives, L-VNIO is the most potent and nNOS-selective reported to date.     

Spectral and cyanide binding properties of the cytochrome aa3 (600 nm) complex from Bacillus subtilis

The cytochrome aa3 (600 nm) complex, or menaquinol oxidase, from Bacillus subtilis is a member of the cytochrome oxidase superfamily of respiratory membrane protein complexes. We have characterized some spectral properties of this enzyme and its reaction with cyanide. The magnetic circular dichroism (MCD) spectrum of the oxidized enzyme has a single band at 1560 nm in the near-infrared region assigned to bis-histidine-ligated, low-spin ferricytochrome a. The other heme, cytochrome a3, is presumably high-spin in the oxidized enzyme, as isolated. The absence of a trough in the MCD spectrum at 790 nm, observed previously with mammalian cytochrome c oxidase and assigned to CuA (Greenwood et al., Biochem. J. 215, 303-316, 1983), is consistent with the absence of this center from the menaquinol oxidase. When the heme ligand cyanide is added to oxidized menaquinol oxidase, a new MCD band appears at 2010 nm, while the band at 1560 nm is unperturbed. The new band is assigned to low-spin ferricytochrome a3 bound with cyanide. The long-wavelength position of this cyanide-induced band is proposed to arise from the close interaction of cytochrome a3 with the copper atom, CuB. The kinetics of cyanide binding to oxidized cytochrome aa3(600 nm) reveal a spectrally simple, yet kinetically complex process. The reaction is biphasic with second-order rate constants of 45 and 0.61 M-1s-1 at 1 mM KCN, with each phase constituting about 50% of the overall reaction. When the enzyme is subjected to a cycle of anaerobic reduction and air oxidation, the subsequent reaction with cyanide occurs in a single phase at the faster rate. This behavior is ascribed to different conformations of the binuclear center exhibiting different reactivities with cyanide, and is in keeping with that previously established for the structurally more complex mitochondrial cytochrome c oxidase. However, the electronic spectral characteristics of some of the species involved in these reactions are different in the present bacterial case from those of reported eukaryotic systems.     

The proximal ligand variant His93Tyr of horse heart myoglobin

The spectroscopic and structural properties of the His93Tyr variant of horse heart myoglobin have been studied to assess the effects of replacing the proximal His residue of this protein with a tyrosyl residue as occurs in catalases from various sources. The variant in the ferric form exhibits electronic spectra that are independent of pH between pH 7 and 10, and it exhibits changes in absorption maxima and intensity that are consistent with a five-coordinate heme iron center at the active site. The EPR spectrum of the variant is that of a high-spin, rhombic system similar to that reported for bovine liver catalase. The 1D 1H-NMR spectrum of the variant confirms the five-coordinate nature of the heme iron center and exhibits a broad resonance at 112.5 ppm that is attributable to the meta protons of the phenolate ligand. This result indicates that the new Tyr ligand flips at a significant rate in this protein. The thermal stability of the Fe(III) derivative is unchanged from that of the wild-type protein (pH 8) while the midpoint reduction potential [-208 mV vs SHE (pH 8.0, 25 degrees C)] is about 250 mV lower. The three-dimensional structure of the variant determined by X-ray diffraction analysis confirms the five-coordinate nature of the heme iron center and establishes that the introduction of a proximal Tyr ligand is accommodated by a shift of the F helix (residues 88-99) in which this residue resides away from the heme pocket. Additional effects of this change are small shifts in the positions of Leu29, a heme propionate, and a heme vinyl group that are accompanied by altered hydrogen bonding interactions with the heme prosthetic group.(ABSTRACT TRUNCATED AT 250 WORDS)     

L-thiocitrulline. A stereospecific, heme-binding inhibitor of nitric-oxide synthases

Nitric-oxide synthase (NOS) catalyzes the oxidation of L-arginine to citrulline and nitric oxide (NO). The enzyme is inhibited by a variety of N omega-monosubstituted L-arginine analogs, and some of these compounds are useful in reversing pathologies associated with the overproduction of NO (e.g. the hypotension of septic shock). We report here that L-thiocitrulline (gamma-thioureido-L-norvaline) is a potent, stereospecific inhibitor of the constitutive brain and endothelial isoforms of NOS as well as the isoform induced in vascular smooth muscle cells by lipopolysaccharide and interferon-gamma. Steady state kinetic studies show L-thiocitrulline inhibition is competitive with L-arginine (Ki approximately 4-20% of KArgm), indicating that initial binding is as a substrate/product analog. In contrast to L-arginine and N omega-methyl-L-arginine, the prototypic NOS inhibitor, L-thiocitrulline binding elicits a "Type II" difference spectrum, indicating a high spin to low spin transition of the iron in the heme cofactor. This finding suggests that L-thiocitrulline is contributing the sixth ligand to heme iron, probably through the thioureido sulfur. Such interaction with heme iron neither stimulates nor inhibits the direct flavin-mediated cytochrome c reduction activity of the enzyme, but it does inhibit heme-dependent superoxide formation. In vivo, L-thiocitrulline is a potent pressor agent in both normal and endotoxemic rats, the latter finding suggesting utility in treating the hypotension of septic shock.     

Cytochrome c peroxidase from Methylococcus capsulatus Bath

A bacterial cytochrome c peroxidase was purified from the obligate methanotroph Methylococcus capsulatus Bath in either the fully oxidized or the half reduced form depending on the purification procedure. The cytochrome was a homo-dimer with a subunit mol mass of 35.8 kDa and an isoelectric point of 4.5. At physiological temperatures, the enzyme contained one high-spin, low-potential (Em7 = -254 mV) and one low-spin, high-potential (Em7 = +432 mM ) heme. The low-potential heme center exhibited a spin-state transition from the penta-coordinated, high-spin configuration to a low-spin configuration upon cooling the enzyme to cryogenic temperatures. Using M. capsulatus Bath ferrocytochrome c555 as the electron donor, the KM and Vmax for peroxide reduction were 510 +/- 100 nM and 425 +/- 22 mol ferrocytochrome c555 oxidized min-1 (mole cytochrome c peroxidase)-1, respectively.     

Intramolecular electron transfer in yeast flavocytochrome b2 upon one-electron photooxidation of the fully reduced enzyme: evidence for redox state control of heme-flavin communication

Flavocytochrome b2, which has been fully reduced using L-lactate, can be rapidly oxidized by 1 equiv using the laser-generated triplet state of 5-deazariboflavin. Parallel photoinduced oxidation occurs at the reduced heme and at the fully reduced FMN (FMNH2) prosthetic groups of different enzyme monomers, producing the anion semiquinone of FMN and a ferric heme. Following the initial oxidation reaction, rapid intramolecular reduction of the ferric heme occurs with concomitant oxidation of FMNH2, generating the neutral FMN semiquinone. The observed rate constant for this intramolecular electron transfer is 2200 s-1, which is 1 order of magnitude larger than the turnover number under these conditions. A slower reduction of the heme prosthetic group also occurs with an observed rate constant of approximately 10 s-1, perhaps due to intersubunit electron transfer from reduced FMN to heme. The rapid intramolecular electron transfer between the FMNH2 and ferric heme is eliminated upon addition of excess pyruvate (Ki = 3.8 mM). This latter result indicates that pyruvate inhibition of catalytic turnover apparently can occur at the FMNH2-->heme electron transfer step. These results markedly differ from those previously obtained (Walker, M. C., & Tollin, G. (1991) Biochemistry 30, 5546-5555) and confirmed here for electron transfer within the one-electron reduced enzyme and for the effect of pyruvate binding, suggesting that intramolecular communication between the heme and flavin prosthetic groups can be controlled by the redox state of the enzyme and by ligand binding to the active site.     

Calmodulin-dependent nitric-oxide synthase. Mechanism of inhibition by imidazole and phenylimidazoles

Calmodulin-dependent nitric-oxide synthase from bovine brain and GH3 pituitary cells is inhibited by imidazole, 1-phenylimidazole, 2-phenylimidazole, and 4-phenylimidazole, with half-maximal inhibition occurring at 200, 25, 160, and 600 microM concentrations of inhibitor, respectively. Imidazole inhibits the maximal velocity of citrulline formation by the enzyme, but does not alter the concentration of arginine, calmodulin, or (6R)-5,6,7,8,-tetrahydro-L-biopterin required for expression of half-maximal activity. Imidazole, 1-phenylimidazole, 2-phenylimidazole, and 4-phenylimidazole had no effect on calmodulin-dependent reduction of cytochrome c by the enzyme at concentrations up to 50-fold higher than those that inhibited citrulline formation. Imidazole inhibited calmodulin-dependent NADPH consumption by the enzyme with dissolved oxygen as the sole electron acceptor, with half-maximal inhibition occurring at a concentration of 225 microM. These observations are consistent with the proposal that imidazole and phenylimidazoles inhibit citrulline formation and oxygen reduction by acting as a sixth coordination ligand of the heme iron. This interaction prevents the formation of the activated reduced species of oxygen necessary for the formation of citrulline.     

Electron transfer in tetrahemic cytochromes c3: spectroelectrochemical evidence for a conformational change triggered by heme IV reduction

Electron transfer in tetrahemic cytochromes c3 from Desulfovibrio vulgaris Hildenborough (D.v.H.) and Desulfovibrio desulfuricans Norway (D.d.N.) strains has been investigated by thin layer spectroelectrochemistry with visible absorption, CD, and resonance Raman (RR) monitoring. The observed splitting of the isosbestic point in the Soret absorption band indicates that the electron transfer from the (FeIII)4 state to the (FeII)4 state proceeds via an intermediate species, which corresponds to 25 and 50% reduction for the D.v.H. cyt.c3 and the D.d.N. cyt.c3, respectively. For the latter, a specific CD signal is observed at half-reduction. RR monitoring of the redox process does not reveal multiple splitting of the high-frequency RR bands, at variance with previously published results on the enzymatic reduction of cyt.c3 from Desulfovibrio vulgaris Miyazaki, a cytochrome highly homologous to D.v.H. cyt.c3 [Verma, A.L., Kimura, A., Nakamura, A., Yagi, T., Inoguchi, H., & Kitagawa, T. (1988) J. Am. Chem. Soc. 110, 6617-6623]. The low-frequency RR spectra of the intermediate species differ significantly from the ones calculated from a linear combination of the all-ferric and all-ferrous states, for the same reduction ratio. Frequency shifts of the bending modes of the cysteine and propionate heme substituents are observed, as well as changes specific to each cytochrome; most notable is the activation of two torsional modes in the case of D.d.N. cyt.c3. Comparison of the results obtained for the two cytochromes leads to the conclusion that reduction of heme IV triggers the observed conformational change. This conclusion is supported by the spectroelectrochemical investigation of the mutant D.v.H. cyt.c3 H25M, in which the sixth ligand of heme III, histidine, is replaced by a methionine.     

Fast cytochrome bo from Escherichia coli binds two molecules of nitric oxide at CuB

The reaction of nitric oxide (NO) with fast cytochrome bo from Escherichia coli has been studied by electronic absorption, MCD, and EPR spectroscopy. Titration of the enzyme with NO showed the formation of two distinct species, consistent with NO binding stoichiometries of 1:1 and 2:1 with observed dissociation constants at pH 7.5 of approximately 2.3 x 10(-)6 and 3.3 x 10(-)5 M. Monitoring the titration by EPR spectroscopy revealed that the broad EPR signals at g approximately 7.3, 3.7, and 2.8 due to magnetic interaction between high-spin heme o (S = 5/2) and CuBII (S = 1/2) are lost. A high-spin heme o signal at g = 6.0 appears as the 1:1 complex is formed but is lost again on formation of the 2:1 complex, which is EPR silent. The absorption spectrum shows that heme o remains in the high-spin FeIII state throughout the titration. These results are consistent with the binding of up to two NO molecules at CuBII. This has been confirmed by studies with the Cl- adduct of fast cytochrome bo. MCD evidence shows that heme o remains ligated by histidine and water. Addition of excess NO to the Cl- adduct leads to the appearance of a high-spin FeIII heme EPR signal. Hence chloride ion binds to CuB, blocking the binding of a second NO molecule. These results suggest a mechanism for the reduction of NO to nitrous oxide by cytochrome bo and cytochrome c oxidase in which the binding of two cis NO molecules at CuB permits the formation of an N-N bond and the abstraction of oxygen by the heme group.     

Effect of excitatory amino acid, dopamine, and oxytocin receptor antagonists on noncontact penile erections and paraventricular nitric oxide production in male rats.

In male rats, noncontact erections occur concomitantly with an increase in NO?- and NO?- in the paraventricular nucleus of the hypothalamus (PVN). In the present study, both responses were reduced by the blockade of PVN excitatory amino acid receptors by dizocilpine, (+)-MK-801(1 and 5 μg), but not by 6-cyano-7-nitro-quinoxaline-2,3-dione (5 μg) or (±)-2-amino-4-phosphono-butanoic acid (5 μg). Also ineffective when injected into the PVN were the dopamine antagonists SCH 23390 (5 μg), S(+)-raclopride (10 μg), and cis-flupenthixol (10 μg), and the oxytocin antagonist d(CH?)?Tyr(Me)2-Orn?-vasotocin (1 μg). However, when the last was given into the lateral ventricles, it reduced noncontact erections without modifying NO?- and NO?- increases. These results suggest that excitatory amino acid transmission increases in the PVN during noncontact erections. This may contribute to increased NO production in the PVN, and it may activate oxytocin neurons mediating this sexual response. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Interaction of peroxynitrite with mitochondrial cytochrome oxidase. Catalytic production of nitric oxide and irreversible inhibition of enzyme activity

Purified mitochondrial cytochrome c oxidase catalyzes the conversion of peroxynitrite to nitric oxide (NO). This reaction is cyanide-sensitive, indicating that the binuclear heme a3/CuB center is the catalytic site. NO production causes a reversible inhibition of turnover, characterized by formation of the cytochrome a3 nitrosyl complex. In addition, peroxynitrite causes irreversible inhibition of cytochrome oxidase, characterized by a decreased Vmax and a raised Km for oxygen. Under these conditions, the redox state of cytochrome a is elevated, indicating inhibition of electron transfer and/or oxygen reduction reactions subsequent to this center. The lipid bilayer is no barrier to these peroxynitrite effects, as NO production and irreversible enzyme inhibition were also observed in cytochrome oxidase proteoliposomes. Addition of 50 microM peroxynitrite to 10 microM fully oxidized enzyme induced spectral changes characteristic of the formation of ferryl cytochrome a3, partial reduction of cytochrome a, and irreversible damage to the CuA site. Higher concentrations of peroxynitrite (250 microM) cause heme degradation. In the fully reduced enzyme, peroxynitrite causes a red shift in the optical spectrum of both cytochromes a and a3, resulting in a symmetrical peak in the visible region. Therefore, peroxynitrite can both modify and degrade the metal centers of cytochrome oxidase.     

Infectious disease complications of simultaneous pancreas kidney transplantation

Gram-positive thermophilic Bacillus species contain cytochrome caa3-type cytochrome c oxidase as their main terminal oxidase in the respiratory chain. To identify alternative oxidases, we isolated several mutants from B. stearothermophilus defective in the caa3-type oxidase activity [Sakamoto, J. et al (1996) FEMS Microbiol. Lett. 143, 151-158]. A novel oxidase was isolated from membrane preparations of one of the mutants, K17. The oxidase was composed of two subunits with molecular masses of 56 and 19 kDa, and contained protoheme IX, heme O, heme A, and Cu in a ratio of 1:0.7:0.2:3. CO difference spectra indicate that the high-spin heme is mainly heme O. These results suggest that the enzyme belongs to the heme-copper oxidase family and is a cytochrome b(o/a)3-type oxidase, whose high-spin heme is mainly heme O and partly heme A. The enzyme oxidized cytochrome c-551, which is a membrane-bound lipoprotein of thermophilic Bacillus. The turnover rate of the activity (Vmax = 190 s[-1]) and its affinity for cytochrome c-551 (Km = 0.15 microM) were much higher than those for yeast and equine heart cytochromes c. The oxidase activity was enhanced by the presence of salts and inhibited by sodium cyanide with a Ki value of 19 microM. The enzyme kinetics suggests that cytochrome c-551 is the natural substrate to this oxidase. Furthermore, the oxidase had similarity to cytochrome ba3-type oxidase from Thermus thermophilus in the subunit composition, partial amino acid sequence, and prosthetic groups, and therefore is suggested to belong to a unique subgroup of the heme-copper oxidase family together with the Thermus enzyme and archaeal oxidases such as Sulfolobus SoxABCD.     

Ligand binding and covalent structure of an oxygen-binding heme protein from Rhodobacter sphaeroides, a representative of a new structural family of c-type cytochromes

The amino acid sequence of an oxygen-binding heme protein (SHP) from Rhodobacter sphaeroides has been determined. The cysteines, which bind the single heme group in the 112-residue protein, are located at positions 43 and 46. SHP is similar in size to the large membrane-bound form of the class I cytochrome c5 of Azotobacter vinelandii (116 residues) and in the location of the heme binding site at positions 48 and 51. Two extra cysteines in SHP (residues 89 and 97) are located in positions similar to those of cytochrome c5 (residues 98 and 101) and form a disulfide bridge in both proteins. In total, four regions of alpha-helix are predicted, covering 46% of the protein, which is comparable to that in other small cytochromes. SHP is thus distantly related to small class I c-type cytochromes but is representative of a distinct family by virtue of its high-spin nature, the lack of a strong sixth ligand, and its capacity to bind oxygen. Potentially, the most important characteristic of SHP is its ability to transiently bind oxygen during autoxidation, which occurs with a half-life of 3 min with a 4-fold excess of O2. SHP also binds carbon monoxide, azide, and cyanide. The kinetics of reduction by free flavins indicate that SHP is less reactive than other class I cytochromes c and that the heme is less accessible to solvent. There is localized positive charge (+3) at the site of reduction of SHP, although the overall protein charge is -2. This may account in part for the ability of SHP to bind anions.     

Temperature-jump and potentiometric studies on recombinant wild type and Y143F and Y254F mutants of Saccharomyces cerevisiae flavocytochrome b2: role of the driving force in intramolecular electron transfer kinetics

The kinetics of intramolecular electron transfer between flavin and heme in Saccharomyces cerevisiae flavocytochrome b2 were investigated by performing potentiometric titrations and temperature-jump experiments on the recombinant wild type and Y143F and Y254F mutants. The midpoint potential of heme was determined by monitoring redox titrations spectrophotometrically, and that of semiquinone flavin/reduced flavin (Fsq/Fred) and oxidized flavin (Fox)/Fsq couples by electron paramagnetic resonance experiments at room temperature. The effects of pyruvate on the kinetic and thermodynamic parameters were also investigated. At room temperature, pH 7.0 and I = 0.1 M, the redox potential of the Fsq/Fred, Fox/Fsq, and oxidized heme/reduced heme (Hox/Hred) couples were -135, -45, and -3 mV, respectively, in the wild-type form. Although neither the mutations nor excess pyruvate did appreciably modify the potential of the heme or that of the Fsq/Fred couple, they led to variable positive shifts in the potential of the Fox/Fsq couple, thus modulating the driving force that characterizes the reduction of heme by the semiquinone in the -42 to +88 mV range. The relaxation rates measured at 16 degreesC in temperature-jump experiments were independent of the protein concentrations, with absorbance changes corresponding to the reduction of the heme. Two relaxation processes were clearly resolved in wild-type flavocytochrome b2 (1/tau1 = 1500 s-1, 1/tau2 = 200 +/- 50 s-1) and were assigned to the reactions whereby the heme is reduced by Fred and Fsq, respectively. The rate of the latter reaction was determined in the whole series of proteins. Its variation as a function of the driving force is well described by the expression obtained from electron-transfer theories, which provides evidence that the intramolecular electron transfer is not controlled by the dynamics of the protein.     

Glutamate 286 in cytochrome aa3 from Rhodobacter sphaeroides is involved in proton uptake during the reaction of the fully-reduced enzyme with dioxygen

The reaction with dioxygen of solubilized fully-reduced wild-type and EQ(I-286) (exchange of glutamate 286 of subunit I for glutamine) mutant cytochrome c oxidase from Rhodobacter sphaeroides has been studied using the flow-flash technique in combination with optical absorption spectroscopy. Proton uptake was measured using a pH-indicator dye. In addition, internal electron-transfer reactions were studied in the absence of oxygen. Glutamate 286 is found in a proton pathway proposed to be used for pumped protons from the crystal structure of cytochrome c oxidase from Paracoccus denitrificans [Iwata et al. (1995) Nature 376, 660-669; E278 in P.d. numbering]. It is the residue closest to the oxygen-binding binuclear center that is clearly a part of the pathway. The results show that the wild-type enzyme becomes fully oxidized in a few milliseconds at pH 7.4 and displays a biphasic proton uptake from the medium. In the EQ(I-286) mutant enzyme, electron transfer after formation of the peroxy intermediate is impaired, CuA remains reduced, and no protons are taken up from the medium. Thus, the results suggest that E(I-286) is necessary for proton uptake after formation of the peroxy intermediate and transfer of the fourth electron to the binuclear center. The results also indicate that the proton uptake associated with formation of the ferryl intermediate controls the electron transfer from CuA to heme a.     

Why practice reduces dual-task interference.

M. A. Van Selst, E. Ruthruff, and J. C. Johnston (1999) found that practice dramatically reduced dual-task interference in a Psychological Refractory Period (PRP) paradigm with 1 vocal response and 1 manual response. Results from 3 further experiments using the highly trained participants of M. A. Van Selst et al. (1999) support 4 main conclusions: (a) A processing bottleneck exists even after extensive practice; (b) the principal cause of the reduction in PRP interference with practice is shortening of Task 1 bottleneck stages; (c) a secondary cause is that 1 or more, but not all, of the Task 2 substages that are postponed before practice are not postponed after practice (i.e., become automatized); and (d) the extent of PRP reduction with practice depends on the modalities of the 2 responses. A control experiment with 2 manual response tasks showed less PRP reduction with practice than that found by Van Selst et al. (PsycINFO Database Record (c) 2010 APA, all rights reserved)