Catechol sensor using poly(aniline-co-o-aminophenol) as an electron transfer mediator

In this work, poly(aniline-co-o-aminophenol) (copolymer) was used as an electron transfer mediator in the electrochemical oxidation of catechol due to its reversible redox over a wide range of pH. The experimental results indicate that the anodic peak potential of catechol at the copolymer electrode is lower than that at the platinum electrode in a solution consisting of catechol and sodium sulfate with pH 5.0, and the activation energy for the electrochemical oxidation of catechol at the copolymer electrode is low (23.6 kJ mol(-1)). These are strong evidence for the electrocatalytic oxidation of catechol at the copolymer electrode. The -OH group on the copolymer chain plays an important role in the electron transfer between the copolymer electrode and catechol in the solution. Based on the catalytic oxidation, the copolymer is used as a sensor to determine the concentration of catechol. The response current of the sensor depends on the concentration of catechol, pH, applied potential and temperature. At 0.55 V (versus saturated calomel reference electrode (SCE)) and pH 5.0, the sensor has a fast response (about 10s) to catechol and good operational stability. The sensor shows a linear response range between 5 and 80 microM catechol with a correlation coefficient of 0.997. It was found that phenol and resorcinol cannot be oxidized at the copolymer electrode at potentials < or =0.55 V, so controlling the sensor potential affords a good way of avoiding the effect of phenol and resorcinol on the determination of catechol.     

Clay-bridged electron transfer between cytochrome p450(cam) and electrode

We demonstrate a very fast heterogeneous redox reaction of substrate-free cytochrome P450(cam) on a glassy carbon electrode modified with sodium montmorillonite. The linear relationship of the peak current in the cyclic voltammogram with the scan rate indicates a reversible one-electron transfer surface process. The electron transfer rate is in the range from 5 to 152 s(-1) with scan rates from 0.4 to 12 V/s, respectively. These values are comparable to rates reported for the natural electron transfer from putidaredoxin to P450(cam). The formal potential of adsorbed P450(cam) is -139 mV (vs NHE) and therefore positively shifted by 164 mV compared to the potential of substrate-free P450(cam) in solution. UV-VIS and FTIR spectra do not indicate an influence of the clay colloidal particles on the heme and the secondary structure of P450(cam) in solution. However, P450(cam) adsorbed on the surface of the clay-modified electrode may undergo partial dehydration resulting in the shift of the formal potential.     

Reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase and cytochrome b5 as electron carriers in NADH-supported cytochrome P-450 -dependent enzyme activities in liver microsomes

The role of NADH-cytochrome b₅ reductase and cytochrome b₅ as electron carriers in NADH-supported electron transport reactions in rat liver microsomes has been examined by measuring three enzyme activities: NADH-cytochrome P-450 reductase, NADH-peroxidase, and NADH-cytochrome c reductase. The first two reactions are known to involve the participation of an NADH-specific reductase and cytochrome P-450 whereas the third requires the reductase and cytochrome b₅. Antibody prepared against NADH-cytochrome b₅ reductase markedly inhibited the NADH-peroxidase and NADH-cytochrome c reductase activities suggesting the involvement of this NADH-specific reductase in these reactions. Liver microsomes prepared from phenobarbital-pretreated rats were digested with subtilisin to remove cytochrome b₅ and the submicrosomal particles were collected by centrifugation. The specific content of cytochrome b₅ in the digested particles was about 5% of that originally present in liver microsomes and all three enzyme activities showed similar decreases whereas NADH-ferricyanide reductase activity (an activity associated with the flavoenzyme NADH-cytochrome b₅ reductase) remained virtually unchanged. Binding of an excess of detergent-purified cytochrome b₅ to the submicrosomal particles at 37 °C for 20 min followed by centrifugation and enzymic measurements revealed a striking increase in the three enzyme activities. Further evidence for cytochrome b₅ involvement in the NADH-peroxidase reaction was the marked inhibition by antibody prepared against the hemoprotein. These results suggest that in microsomal NADH-supported cytochrome P-450-dependent electron transport reactions, cytochrome b₅ functions as an intermediate electron carrier between NADH-cytochrome b₅ reductase and cytochrome P-450.     

Thermodynamically controlled supramolecular polymerization of cytochrome b562

A hemoprotein‐based supramolecular polymer that has a covalently linked heme moiety on the protein surface has been constructed based on interprotein heme–heme pocket interactions of the chemically modified apocytochrome b₅₆₂ ( 1 ‐H63C). The thermodynamic properties of the polymer have been investigated by means of size exclusion chromatography, UV–vis spectroscopy, and circular dichroism spectroscopy. The results indicate that, as with other synthetic systems reported so far, the 1 ‐H63C hemoprotein assembly is thermodynamically controlled in aqueous solution: the degree of polymerization is dependent on the 1 ‐H63C concentration and is modulated by the addition of the end‐capping units, native heme, and/or apocytochrome b₅₆₂ mutant (apoH63C). These properties suggest a potential use for the hemoprotein self‐assembly in preparation of stimuli‐responsive functional nanobiomaterials. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 194–200, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Characterisation of a Rrhodobacter sphaeroides gene that encodes a product resembling Eescherichia coli cytochrome b(561) and R. sphaeroides cytochrome b(562)

Analysis of the photoactive yellow protein (pyp) gene region of Rhodobacter sphaeroides has revealed the presence of an additional open reading frame, orfD, that had not previously been identified. Here we report the location of this new gene and the predicted amino acid sequence of the encoded protein. The translation product resembles a group of small cytochrome b-like proteins, including Escherichia coli cytochrome b(561), R. sphaeroides cytochrome b(562), and two new cytochrome b(561)-like proteins identified using the E. coli genome sequence, for which functions have not yet been established. To determine OrfD function in R. sphaeroides, an orfD mutant was constructed. The OrfD mutant exhibited growth rates and yields very similar to those of the wild-type strain when grown under a variety of growth conditions. Respiration rates, reduced-minus-oxidised spectra and levels of photosynthetic complexes were also very similar in the two strains. Although the role of OrfD was therefore not determined here, we demonstrate that the orfD gene is expressed in R. sphaeroides under aerobic, semi-aerobic and photosynthetic growth conditions.     

Design of an artificial light-harvesting unit by protein engineering: cytochrome b(562)-green fluorescent Protein chimera.

We have generated a novel model protein for an artificial light-harvesting complex composed of two proteins, cytochrome b(562) (cytb(562)) and enhanced green fluorescent protein (EGFP), in which two chromophores are fixed in each protein matrix. Cytb(562) was appended to the N-terminus of EGFP via a Gly-Ser linker and the resultant fusion protein was successfully expressed in Escherichia coli as a mixture of the apo- and the holo-forms as to the cytb(562) moiety. The fluorescence of EGFP was substantially quenched when the apo-form was reconstituted with hemin. Based on the fluorescence lifetime measurements, it appeared that light energy entrapped by EGFP is transferred to the heme of cytb(562) by resonance energy transfer (energy transfer yield: 65%). Spatial organization of two chromophores using small and stable protein matrices will be promising toward the construction of an artificial light-harvesting complex by protein engineering.     

Crystallographic study of cytochrome b 562 from Escherichia coli

Three crystalline forms of cytochrome b₅₆₂ from Escherichia coli have been grown. One form, suitable for a three-dimensional structure investigation, is triclinic, containing two molecules per cell, with the following parameters: a = 33·71 Å, b = 50·51 Å, c = 32·71 Å, α = 102.62°, β = 86.67° and γ = 107.03°. X-ray data from the native crystals have been collected to 2.8 Å resolution and a search for suitable heavy atom derivatives has begun.     

Conversion of the Escherichia coli cytochrome b562 to an archetype cytochrome b: a mutant with bis-histidine ligation of heme iron

A mutant of the Escherichia coli cytochrome b(562) has been created in which the heme-ligating methionine (Met) at position 7 has been replaced with a histidine (His) (M7H). This protein is a double mutant that also has the His 63 to asparagine (H63N) mutation, which removes a solvent-exposed His. While the H63N mutation has no measurable effect on the cytochrome, the M7H mutation converts the atypical His/Met heme ligation in cytochrome b(562) to the classic cytochrome b-type bis-His ligation. This mutation has little effect on the K(d) of heme binding but significantly reduces the chemical and thermal stability of the mutant cytochrome relative to the wild type (wt). Both proteins have similar absorbance (Abs) and electron paramagnetic resonance (EPR) properties characteristic of 6-coordinate low-spin heme. The Abs spectra of the oxidized and reduced bis-His cytochrome are slightly blue-shifted relative to the wt, and the alpha Abs band of ferrous M7H mutant is unusually split. The M7H mutation decreases the midpoint potential of the bound heme by 260 mV at pH 7 and considerably alters the pH dependence of the E(m), which becomes dominated by a single pK(red) = 6.8.     

Determination of kinase activity by using an enzyme electrode

A method for determining the enzymatic activity of hexokinases, acetate kinase and pyruvate kinase using an enzyme electrode was developed. The assay time is 2-3 min. The lower limit of the activity determining is 0,054 U/ml. The proposed method was applied to investigation of pyruvate kinase and acetate kinase reactivation under the action of mercaptoethanol.     

A multigeneration analysis of cytochrome b(562) redox variants: evolutionary strategies for modulating redox potential revealed using a library approach

The redox potential of cytochromes sets the energy yield possible in metabolism and is also a key determinant of the rate at which redox reactions proceed. Here, the heme protein, cytochrome b(562), is used to study the in vitro evolution of redox potential within a library of variants containing the same structural archetype, the four-helix bundle. Multisite variations in the active site of cytochrome b(562) were introduced. A library of variants containing random mutations in place of R98 and R106 was created, and the redox potentials of a statistical sampling of this library were measured. This procedure was carried out for both the low- and high-potential variants of a previously studied F61X/F65X, first-generation library [Springs, S. L., Bass, S. E., and McLendon, G. L. (2000) Biochemistry 39, 6075]. The second-generation library reported here has a range of redox potentials which is greater than 40% (160 mV) of the known accessible potential among cytochromes with identical axial ligands (but different folds) and exceeds the range exhibited phylogenetically by the cytochrome c' family which internally maintains the same axial ligation and fold. A statistical analysis of the libraries examined reveals that the redox potential of WT cyt b(562) is found at the high-potential extremum of the distribution, indicating that this protein apparently evolved to differentially stabilize the reduced protein. The 2.7 A crystal structure of F61I/F65Y/R106L (low-potential variant of the second-generation library) was solved and is compared to the wild-type structure and the 2.2 A resolution structure of the F61I/F65Y variant (low-potential variant of the first-generation library). The structures indicate that charge-dipole effects are responsible for shifting the redox equilibrium toward the oxidized state in both the F61I/F65Y and F61I/F65Y/R106L variants. Specifically, a new protein dipole is introduced into the heme microenvironment as a result of the F65Y mutation, two new internal water molecules (one in hydrogen-bonding distance of Y65) are found, and in the case of F61I/F65Y/R106L (DeltaE(m) = 158 mV vs NHE), increased solvent exposure of the heme as a result of the R106L substitution is identified.     

Domain movement of iron sulfur protein in cytochrome bc1 complex is facilitated by the electron transfer from cytochrome b(L) to b(H)

The key step of the "protonmotive Q-cycle" mechanism for cytochrome bc1 complex is the bifurcated oxidation of ubiquinol at the Qp site. ISP is reduced when its head domain is at the b-position and subsequent move to the c1 position, to reduce cytochrome c1, upon protein conformational changes caused by the electron transfer from cytochrome b(L) to b(H). Results of analyses of the inhibitory efficacy and the binding affinity, determined by isothermal titration calorimetry, of Pm and Pf, on different redox states of cytochrome bc1 complexes, confirm this speculation. Pm inhibitor has a higher affinity and better efficacy with the cytochrome b(H) reduced complex and Pf binds better and has a higher efficacy with the ISP reduced complex.     

A cytochrome b562 variant with a c-type cytochrome CXXCH heme-binding motif as a probe of the Escherichia coli cytochrome c maturation system

Cytochrome b562 is a periplasmic Escherichia coli protein; previous work has shown that heme can be attached covalently in vivo as a consequence of introduction of one or two cysteines into the heme-binding pocket. A heterogeneous mixture of products was obtained, and it was not established whether the covalent bond formation was catalyzed or spontaneous. Here, we show that coexpression from plasmids of a variant of cytochrome b562 containing a CXXCH heme-binding motif with the E. coli cytochrome c maturation (Ccm) proteins results in an essentially homogeneous product that is a correctly matured c-type cytochrome. Formation of the holocytochrome was accompanied by substantial production of its apo form, in which, for the protein as isolated, there is a disulfide bond between the two cysteines in the CXXCH motif. Following addition of heme to reduced CXXCH apoprotein, spontaneous covalent addition of heme to polypeptide occurred in vitro. Strikingly, the spectral properties were very similar to those of the material obtained from cells in which presumed uncatalyzed addition of heme (i.e. in the absence of Ccm) had been observed. The major product from uncatalyzed heme attachment was an incorrectly matured cytochrome with the heme rotated by 180 degrees relative to its normal orientation. The contrast between Ccm-dependent and Ccm-independent covalent attachment of heme indicates that the Ccm apparatus presents heme to the protein only in the orientation that results in formation of the correct product and also that heme does not become covalently attached to the apocytochrome b562 CXXCH variant without being handled by the Ccm system in the periplasm. The CXXCH variant of cytochrome b562 was also expressed in E. coli strains deficient in the periplasmic reductant DsbD or oxidant DsbA. In the DsbA- strain under aerobic conditions, c-type cytochromes were made abundantly and correctly when the Ccm proteins were expressed. This contrasts with previous reports indicating that DsbA is essential for cytochrome c biogenesis in E. coli.     

The solution structure of oxidized Escherichia coli cytochrome b562.

The solution structure of the oxidized, paramagnetic form of cytochrome b562 from Escherichia coli (106 amino acids) is here reported as obtained from 1653 meaningful NOEs (from a total of 2051 unique NOEs), 33 (3)JHNHalpha values, and 339 pseudocontact shifts. The structure displays the typical four-helix bundle motif, and a disordered loop between helices alpha2 and alpha3, as found in the solid state. The solution structure has a conformation intermediate between the two independent solid-state molecules, although different orientations are observed for a few residues. The magnetic susceptibility tensor is similar to that of cytochrome c, which has the same ligands, although the anisotropy is somewhat smaller. This difference in the electronic structure is consistent with the thermal accessibility in cytochrome b562 of states with S > 1/2. The structure is also compared with the solution structure of the apoprotein, and some information on the role of the cofactor on the protein folding and mobility is obtained. Helix alpha4 seems to be the most sensitive to the chemical environment in terms of structure and mobility. The pKa values affecting the hyperfine-shifted signals are also discussed. Quite intriguing is the comparison of the structure of cytochrome b562 with the available structures of cytochromes c' which display a similar folding motif and similar pKa values but very little sequence similarity.     

Localization of the redox-center of cytochrome b562 on the internal (M-) side of the mitochondrial membrane

In the inside-out submitochondrial particles, cytochrome b-562 is readily reduced by a hydrophilic redox mediator Ru(NH3)6(2)+; this reaction is not inhibited by antimycin and myxothiazol. In mitochondria, cytochromes b do not virtually interact with Ru(NH3)6(2)+. The accessibility of cytochrome b-562 to Ru(NH3)6(2)+ in submitochondrial particles and its inaccessibility in mitochondria suggest the localization of the hemoprotein redox center on the inner surface of the mitochondrial membrane.     

Fabrication of polymeric electron-transfer mediator/enzyme hydrogel multilayer on an Au electrode in a layer-by-layer process

The layer-by-layer (LBL) construction of an enzyme electrode covered with a multilayer structure alternately composed of a polymeric electron transfer mediator and a polymer-modified enzyme was examined. Poly(2-methacryloyloxyethyl phosphorylcholine-co-p-vinylphenylboronic acid-co-vinylferrocene) (PMVF) was synthesized and used as a polymeric electron transfer mediator. Glucose oxidase (GOx) was selected as a model enzyme and poly(vinyl alcohol) (PVA) chains were bound to the GOx (GOx-PVA) under mild conditions. The PMVF and PVA formed a gel spontaneously through a selective reaction between phenylboronic acid units and hydroxyl groups in both polymers. Using the spin coating technique, a repeating PMVF/GOx-PVA multilayer was fabricated on the surface of an Au electrode. The thickness of each PMVF/GOx-PVA layer was around 5.8 nm, corresponding to the dimensions of GOx. The electrochemical performance of the electrode was evaluated in glucose concentration measurement. The oxidation current of glucose by GOx was measured at 0.38 V (vs. Ag/AgCl), verifying that ferrocene units in the PMVF of the hydrogel electrically wired the immobilized GOx. Moreover, the current increased with the number of PMVF/GOx-PVA layers. That is, both intermolecular electron transfer between each individual layer and the presence of a freely diffusing substrate in the hydrogel were achieved. We conclude that a LBL structure constructed from PMVF and a PVA-modified enzyme is effective for use in developing bioelectronic devices that employ enzyme molecules.     

Mapping the electron transfer interface between cytochrome b5 and cytochrome c

To characterize the cytochrome b(5) (Cyt b(5))-cytochrome c (Cyt c) interactions during electron transfer, variants of Cyt b(5) have been employed to assess the contributions of electrostatic interactions (substitution of surface charged residues Glu44, Glu48, Glu56, and Asp60 and heme propionate), hydrophobic interactions, and the thermodynamic driving forces (substitutions for hydrophobic residues in heme pocket residues Phe35, Pro40, Val45, Phe58, and Val61). The electrostatic interactions play an important role in maintaining the stability and specificity of the Cyt b(5)-Cyt c complex that is formed. There is no essential effect on the intraprotein complex electron transfer even if most of the involved negatively charged residues on the surface of Cyt b(5) have been removed. The results support a dynamic docking paradigm for Cyt b(5)-Cyt c interactions. The orientation that is optimal for binding may not be optimal form for electron transfer. Substitution of hydrophobic residues does not have a significant effect on the binding between Cyt b(5) and Cyt c; rather, it regulates the electron transfer rates via changes in the driving force. Combining the electron transfer studies of the Cyt b(5)-Cyt c system and the Cyt b(5)-Zn-Cyt c system, we obtain the reorganization energy (0.6 eV) at an ionic strength of 150 mM.