Direct electrochemistry of cytochrome c at ordered macroporous active carbon electrode

Three-dimensionally (3D) ordered macroporous active carbon has been fabricated and used as electrode substrate for the direct electrochemistry of horse heart cytochrome c (Cyt c). The Cyt c immobilized on the surface of the ordered macroporous active carbon shows a pair of well-defined and nearly reversible redox waves at the formal potential of −0.033 V in pH 6.8 phosphate buffer solution. The interaction between Cyt c and the 3D macroporous active carbon makes the formal potential shift negatively compared to that of Cyt c in solution. Spectrophotometric and electrochemical methods have been used to investigate the interaction between Cyt c and the porous active carbon. The immobilized Cyt c maintains its biological activity, and shows a surface controlled electrode process with the electron-transfer rate constant (k_s) of 17.6 s⁻¹ and the charge-transfer coefficient (a) of 0.52, and displays the features of a peroxidase in the electrocatalytic reduction of hydrogen peroxide (H₂O₂). A potential application of the Cyt c-immobilized porous carbon electrode as a biosensor to monitor H₂O₂ has been investigated. The steady-state current response increases linearly with H₂O₂ concentration from 2.0 × 10⁻⁵ to 2.4 × 10⁻⁴ mol l⁻¹. The detection limit (3σ) for determination of H₂O₂ has been found to be 1.46 × 10⁻⁵ mol l⁻¹.     

Direct electrochemistry of protein-protein complexes involving cytochrome c, cytochrome b5, and plastocyanin

The direct electrochemistry of the cytochrome c/cytochrome b5 and cytochrome c/plastocyanin complexes has been investigated at edge-plane graphite and modified gold electrode surfaces, which are selective for one of the two components of the complex. Electrochemical response of one protein at an otherwise electrostatically unfavorable electrode surface was achieved in the presence of the other protein, and the calculated heterogeneous electron-transfer rate constant and diffusion coefficient were found to be in good agreement with the values determined previously from the electrochemistry of the individual proteins [Armstrong, F. A., Hill, H. A. O., & Walton, N. J. (1988) Acc. Chem. Res. 21, 407 and references therein]. A dynamic model of the protein-protein-electrode ternary complex is proposed to explain the promotion effect, and this model is supported by a study comparing the electrochemical responses of covalent and electrostatic cytochrome c/plastocyanin complexes. It is also suggested that the behavior of protein-protein complexes at electrode surfaces could be related to that of the complexes associated with biological membranes.     

Construction and direct electrochemistry of orientation controlled laccase electrode

A laccase has multiple redox centres. Chemisorption of laccases on a gold electrode through a polypeptide tag introduced at the protein surface provides an isotropic orientation of laccases on the Au surface, which allows the orientation dependent study of the direct electrochemistry of laccase. In this paper, using genetic engineering technology, two forms of recombinant laccase which has Cys-6×His tag at the N or C terminus were generated. Via the Au-S linkage, the recombinant laccase was assembled orientationally on gold electrode. A direct electron transfer and a bioelectrocatalytic activity toward oxygen reduction were observed on the two orientation controlled laccase electrodes, but their electrochemical behaviors were found to be quite different. The orientation of laccase on the gold electrode affects both the electron transfer pathway and the electron transfer efficiency of O₂ reduction. The present study is helpful not only to the in-depth understanding of the direct electrochemistry of laccase, but also to the development of laccase-based biofuel cells.     

Syntheses of fully sulfonated polyaniline nano-networks and its application to the direct electrochemistry of cytochrome c

Fully sulfonated polyaniline nano-particles, nano-fibrils and nano-networks have been achieved for the first time by electrochemical homopolymerization of orthanilic acid using a three-step electrochemical deposition procedure in a mixed solvent of acetonitrile (ACN) and water. The diameter of the uniform nano-particles is about 60 nm, and the nano-fibrils can be organized in two-dimensional (2D) or three-dimensional (3D) non-periodic networks with good electrical contact. Average distance between contacts is about 850 and 600nm for a 2D and 3D system, respectively. The details of the poly(orthanilic acid) (POA) nano-structure were examined with a field emission scanning electron microscope (SEM). The structure and properties of POA were characterized with FTIR, UV-vis and electrochemical methods. The 3D POA nano-networks coated platinum electrode gave a direct electrochemical behavior of horse heart cytochrome c (Cyt c) immobilized on this electrode surface, a pair of well-defined redox waves with formal potential (E( degrees ')) of -0.032 V (versus Ag/AgCl) was achieved. The interaction between Cyt c and POA makes the formal potential shift negatively compared to that of Cyt c in solution. Spectrophotometric and electrochemical methods were used to investigate the interaction of Cyt c with POA. The immobilized Cyt c in the nano-networks POA film maintained its activity, showing a surface-controlled electrode process with the electron transfer rate constant (k(s)) of 21s(-1) and a of 0.53, and could be used for the electrocatalytic reduction of hydrogen peroxide. The quantitative determination of Cyt c by differential pulse voltammetry (DPV) using the fully sulfonated 3D POA nano-networks film coated platinum electrode was also studied.     

Direct electrochemistry of proteins. Investigations of yeast cytochrome c mutants and their complexes with cytochrome b5.

Direct electrochemistry of site-specific mutants of yeast iso-1-cytochrome c (cyt c) and their complexes with bovine cytochrome b5 (cyt b5) has been investigated at edge-plane pyrolytic graphite (EPG) and bis(4-pyridyl)-disulphide-modified gold electrodes. Structure/function relationships have been investigated with the particular aim of clarifying the factors controlling the interactions of proteins at electrode/electrolyte interfaces and the determinants for direct electrochemistry in ternary protein/protein/electrode adducts, e.g. cyt c/cyt b5/EPG. Investigations of the cyt c mutants alone revealed a variety of electrochemical responses: all the mutants show similar voltammetric reversibility at modified gold electrodes, whereas at EPG electrodes the reversibility follows the order: Asn52Ile-Cys102Thr greater than Cys102Thr greater than Asn52Ala-Cys102Thr. Mid-point potentials follow the order: Arg13Ile (+60 +/- 5 mV vs. standard calomel electrode) greater than Cys102Thr (+40 +/- 5 mV) greater than Lys27Gln (+30 +/- 5 mV) approximately Lys72Asp (+30 +/- 5 mV) greater than Asn52Ala-Cys102Thr (+15 +/- 5 mV) greater than Asn52Ile-Cys102Thr (-10 +/- 5 mV). The structural basis for these differences is briefly discussed. When these mutants are bound to cyt b5, the differences in electrochemical response are greatly enhanced in the ternary cyt c/cyt b5/EPG adducts. A minimal analysis of these differences supports a model of multiple overlapping binding and recognition domains on cyt c which may be finely tuned to allow ternary complex formation so that a single-site variation could modify or abolish direct electrochemistry in the ternary adduct.     

Mechanism of inactivation of gamma-aminobutyric acid aminotransferase by (S,E)-4-amino-5-fluoropent-2-enoic acid

Evidence for an enamine mechanism of inactivation of pig brain gamma-aminobutyric acid (GABA) aminotransferase by (S,E)-4-amino-5-fluoropent-2-enoic acid is presented. apo-GABA aminotransferase reconstituted with [3H]pyridoxal 5'-phosphate is inactivated by (S,E)-4-amino-5-fluoropent-2-enoic acid and the pH is raised to 12. All of the radioactivity is released from the enzyme as an adduct of the cofactor; no [3H]pyridoxamine 5'-phosphate is generated.     

Stabilization of Pseudomonas aeruginosa cytochrome c(551) by systematic amino acid substitutions based on the structure of thermophilic Hydrogenobacter thermophilus cytochrome c(552)

A heterologous overexpression system for mesophilic Pseudomonas aeruginosa holocytochrome c(551) (PA c(551)) was established using Escherichia coli as a host organism. Amino acid residues were systematically substituted in three regions of PA c(551) with the corresponding residues from thermophilic Hydrogenobacter thermophilus cytochrome c(552) (HT c(552)), which has similar main chain folding to PA c(551), but is more stable to heat. Thermodynamic properties of PA c(551) with one of three single mutations (Phe-7 to Ala, Phe-34 to Tyr, or Val-78 to Ile) showed that these mutants had increased thermostability compared with that of the wild-type. Ala-7 and Ile-78 may contribute to the thermostability by tighter hydrophobic packing, which is indicated by the three dimensional structure comparison of PA c(551) with HT c(552). In the Phe-34 to Tyr mutant, the hydroxyl group of the Tyr residue and the guanidyl base of Arg-47 formed a hydrogen bond, which did not exist between the corresponding residues in HT c(552). We also found that stability of mutant proteins to denaturation by guanidine hydrochloride correlated with that against the thermal denaturation. These results and others described here suggest that significant stabilization of PA c(551) can be achieved through a few amino acid substitutions determined by molecular modeling with reference to the structure of HT c(552). The higher stability of HT c(552) may in part be attributed to some of these substitutions.     

Effect of adenosine 5'-triphosphate and adenosine 5'-diphosphate on the oxidation of cytochrome c by cytochrome c oxidase

Adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and inorganic pyrophosphate partially inhibit the oxidation of exogenous cytochrome c by cytochrome c oxidase of submitochondrial particles (with or without detergent treatment) or by a purified preparation when it is assayed polarographically in buffers of nonbinding ions at pH 7.8. ATP is somewhat more inhibitory than ADP. The inhibition is never greater than 50%, and it is always less than an equal concentration of Mg2+ ions is present or when the assays are run at pH 6. In contrast, the effect of ATP, ADP, and pyrophosphate on oxidase assays run spectrophotometrically is a similar slight stimulation of the oxidase of submitochondrial particles treated with deoxycholate and little or no effect on purified oxidase. The reaction of the oxidase of submitochondrial particles with the endogenous cytochrome c is stimulated by the nucleotides, as is the reduced nicotinamide adenine dinucleotide (NADH) oxidase activity. The observations can be explained by binding of ATP, ADP, or pyrophosphate to cytochrome c so that the formation of an especially reactive combination of cytochrome c and cytochrome oxidase previously postulated [Smith, L., Davies, H. C., & Nava, M. E. (1979) Biochemistry 18, 3140] is prevented. The data give no evidence that respiration via cytochrome c oxidase is regulated physiologically by direct effects of ATP or ADP on its activity.     

Urchinlike MnO2 nanoparticles for the direct electrochemistry of hemoglobin with carbon ionic liquid electrode

In this paper an urchinlike MnO(2) nanoparticle was synthesized by hydrothermal method and applied to the protein electrochemistry for the first time. By using a carbon ionic liquid electrode (CILE) as the basal electrode, hemoglobin (Hb) was immobilized on the surface of CILE with chitosan (CTS) and MnO(2) nanoparticle composite materials. Spectroscopic results indicated that Hb molecules retained its native structure in the composite film. A pair of well-defined redox peaks appeared on the cyclic voltammogram with the formal peak potential as -0.180 V (vs. SCE), which indicated that direct electron transfer of Hb was realized on the modified electrode. The result can be attributed to the specific characteristic of MnO(2) nanoparticle and the advantages of CILE, which facilitated the electron transfer rate. The fabricated CTS-MnO(2)-Hb/CILE showed good electrocatalytic ability to the reduction of trichloroacetic acid (TCA). Under the optimal conditions the catalytic current was in linear to TCA concentration in the range from 0.5 to 16.0 mmol L(-1) with the detection limit calculated as 0.167 mmol L(-1) (3σ). The result indicated that urchinlike MnO(2) nanoparticle had the potential application in the third generation electrochemical biosensors.     

Metal-mediated reduction of cytochrome c by thioglycolic acid

The reduction of cytochrome c by thioglycolic acid was found to be extremely sensitive to metal catalysis. The rate of the uncatalyzed reaction was negligible in comparison with rates obtained from reactions supplemented with catalytic amounts of copper or iron. Both the catalyzed and uncatalyzed reactions were independent of pH (near neutrality) but when o-phenanthroline was included in the reaction mixture, a pH dependence was induced. This pH dependence is the result of an interference of oxygen with the metal complexes. A comparison of the rate constants at zero ionic strenght, which were obtained from the application of the Debye-Hückel theory for the ionic strength dependence, demonstrated that copper complexes are superior catalysts as compared with iron complexes. Our results suggest that in the copper-mediated reaction, the catalyst is a cupric thioglycolate complex with a net charge of ?2. The addition of o-phenanthroline to the reaction mixture results in a tenfold decrease in the catalytic activity and in a change in the net charge of the catalyst to ?1. At pH 8 the iron-mediated reduction is catalyzed by a ferric thioglycolate complex, whereas at pH 7 a ferrothioglycolate complex provides the catalytic activity. Both complexes have a net negative charge of ?2. At both pH's the catalytic activity is completely abolished by the addition of o-phenanthroline. The results demonstrate the effectiveness by which metal-sulfur complexes can facilitate one-electron transfer reactions and could there-fore serve as a model in the study of various biological oxidations.     

Isolation and identification of (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid from anAmanita of the sectionRoanokenses (Amanitaceae)

A chlorine-containing non-protein amino acid which was recently discovered from the fruit bodies ofAmanita gymnopus (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid, was isolated and crystallized for the first time from the fruit bodies of an unknown member ofAmanita belonging to the sectionRoanokenses, subsectionSolitariae. The results of elementary analyses, determination of optical rotations,¹H- and¹³C-NMR-spectra, and some chemical reactions supported an earlier proposed structure.Part 24 in the series Biochemical studies of nitrogen compounds in fungi. for Part 23, see Hatanaka, S. I. et al. 1994. this journal35: 391–394.     

The rate-limiting step in O(2) reduction by cytochrome ba(3) from Thermus thermophilus

Cytochrome ba(3) (ba(3)) of Thermus thermophilus (T. thermophilus) is a member of the heme-copper oxidase family, which has a binuclear catalytic center comprised of a heme (heme a(3)) and a copper (Cu(B)). The heme-copper oxidases generally catalyze the four electron reduction of molecular oxygen in a sequence involving several intermediates. We have investigated the reaction of the fully reduced ba(3) with O(2) using stopped-flow techniques. Transient visible absorption spectra indicated that a fraction of the enzyme decayed to the oxidized state within the dead time (~1ms) of the stopped-flow instrument, while the remaining amount was in a reduced state that decayed slowly (k=400s(-1)) to the oxidized state without accumulation of detectable intermediates. Furthermore, no accumulation of intermediate species at 1ms was detected in time resolved resonance Raman measurements of the reaction. These findings suggest that O(2) binds rapidly to heme a(3) in one fraction of the enzyme and progresses to the oxidized state. In the other fraction of the enzyme, O(2) binds transiently to a trap, likely Cu(B), prior to its migration to heme a(3) for the oxidative reaction, highlighting the critical role of Cu(B) in regulating the oxygen reaction kinetics in the oxidase superfamily.     

The rate-limiting step and nonhyperbolic kinetics in the oxidation of ferrocytochrome c catalyzed by cytochrome c oxidase

The level of reduction of cytochrome a and CuA during the oxidation of ferrocytochrome c has been determined in stopped-flow experiments. Both components are partially reduced but become progressively more oxidized as the reaction proceeds. When all cytochrome c has been oxidized, CuA is also completely oxidized, whereas cytochrome a is still partially reduced. These results can be simulated on the basis of a model which requires that the intramolecular electron transfer from cytochrome a and CuA to cytochrome a3-CuB is a two-electron process and, in addition, that the binding of oxidized cytochrome c to the electron- transfer site decreases the rate constants for intramolecular electron transfer from cytochrome a. The first requirement is related to the function of the oxidase as a proton pump. Product dissociation is not by itself rate-limiting, making it less likely that the source of the nonhyperbolic substrate kinetics is an effect on this step from electrostatic interaction with ferricytochrome c bound to a second site. It is pointed out that nonhyperbolic kinetics is, in fact, an intrinsic property of ion pumps.     

Distribution of ciliatine (2-aminoethylphosphonic acid) and phosphonoalanine (2-amino-3-phosphonopropionic acid) in human tissues

Ciliatine (2-aminoethylphosphonic acid) was detected in the human brain, heart, kidney, liver, intestine, spleen, adrenal glands, and aorta. Phosphonoalanine (2-amino-3-phosphonopropionic acid) was found in the human liver, intestine and spleen. Tissue homogenates were extracted with trichloroacetic acid and a chloroform-methanol mixture. After hydrolysis, each fraction was subfractionated by ion-exchange chromatography and examined by paper chromatography and electrophoresis using a specific ninhydrin-molybdate staining procedure to detect the phosphonic acids. The acids were found bound either to lipid or to protein; no free phosphonic acid was detected.     

Enantioselective actions of 4-amino-3-hydroxybutanoic acid and (3-amino-2-hydroxypropyl)methylphosphinic acid at recombinant GABA(C) receptors

The R- and S-enantiomers of 4-amino-3-hydroxybutanoic acid (GABOB) were full agonists at human recombinant rho1 GABA(C) receptors. Their enantioselectivity (R>S) matched that reported for their agonist actions at GABA(B) receptors, but was the opposite to that reported at GABA(A) receptors (S>R). The corresponding methylphosphinic acid analogues proved to be rho1 GABA(C) receptor antagonists with R(+)-CGP44533 being more potent than S(-)-CGP44532, thus showing the opposite enantioselectivity to the agonists R(-)- and S(+)-GABOB. These studies highlight the different stereochemical requirements for the hydroxy group in these analogues at GABA(A), GABA(B) and GABA(C) receptors.     

2-amino-4-acetylaminobutyric acid, 2,4-diaminobutyric acid and 2-amino-6N-oxalylureidopropionic acid (oxalylalbizziine) in seeds of acacia angustissima

A new amino acid previously detected in 17 species of Acacia has been isolated from seeds of Acacia angustissima and identified as oxalylalbizziine. These seeds also contain more than 6% dry weight of 2-amino-4-acetylaminobutyric acid, which has not been reported previously in a legume, and lower concentrations of 2,4-diaminobutyric acid.     

Synthesis and antibacterial activities of 5-hydroxy-4-amino-2(5H)-furanones

Starting from the mucohalogen acids 1a and b 5-hydroxy-2(5H)-furanones 2a-h have been prepared and tested. These novel 4-amino-5-hydroxy 2(5H)-furananones have shown a broad antibiotic activity against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 in the micromolar range. A one step synthesis from mucohalogen acids towards the antibacterials 2a-h was developed, in which the target was obtained from 1a and b under reflux in toluene in presence of a catalytic amount of sulfuric acid. The derivatives 2b and c displayed a MIC and MBC of 4/8mug/ml, against Staphylococcus aureus with a selectivity towards the resistant strains.