光合细菌Chromatium vinosum可溶性氢酶的EPR研究

光合细菌 Chromatium vinosum可溶性氢酶经 5次柱层析 ( DE-2 3,TSK-DEAE( ) ,Ultragel Ac A-4 4 ,TSK-DEAE( ) ,Superdex TM75 )分离后被纯化 365倍 ,得率为 32 % ,其催化放氢的活性为 8.4μmol H2 /( min· mg prot) .氧化态可溶性氢酶在 4 5 K下 ,产生了 Ni( )的典型 EPR信号 ( gx,y,z=2 .37,2 .1 6,2 .0 1 6和 gx,y,z=2 .30 ,2 .2 3,2 .0 1 6) ;在 1 0 K下 ,没有出现膜结合态氢酶中存在的 [3Fe-4 S]簇特征 EPR信号 .可溶性氢酶被 H2 还原后 ,Ni( )的特征 EPR信号消失 ,同时出现一个 [4 Fe-4 S]簇的特征 EPR信号 ( gx,y,z=1 .88,1 .90 ,2 .0 4 5 ) .研究结果表明 ,C.vinosum可溶性氢酶在结构和功能上不同于膜结合态氢酶 ,是一种新的催化放氢的 Ni Fe-氢酶     

Studies on Cytochrome c Modified by [PtdienNO_3]Cl

For the first time, [PtdienNO_3]Cl was used as a stable reagent to modify ferricytochrome c and the reaction products were separated and purified with the CM-52 cation exchange chromatography. Five components were obtained, corresponding to the native cytochrome c single-labeled, dual-labeled, and triple-labeled derivatives as shown by the analysis of the molar ratio of the two metal atoms (Pt and Fe). The reduction potentials of these proteins were measured by differential pulse voltammetry. His-33 and Trp-59 were identified by~1HNMR as the binding sites of the platinum complex in the modified cytochrome c derivatives. Trp-59 was a conserved amino acid connected with the heme through hydrogen bond, which had not been modified by other transition metal complexes. The platinummodified cytochrome c derivatives might be valuable in exploring the role of the aromatic amino acids, especially Trp-59, in electron transfer.     

Direct electrochemistry behavior of Cytochrome c on silicon dioxide nanoparticles-modified electrode

A newfangled direct electrochemistry behavior of Cytochrome c (Cyt c) was found on glassy carbon (GC) electrode modified with the silicon dioxide (SiO2) nanoparticles by physical adsorption. A pair of stable and well-defined redox peaks of Cyt c′ quasi-reversible electrochemical reaction were obtained with a heterogeneous electron transfer rate constant of 1.66×10-3 cm/s and a formal potential of 0.069 V (vs. Ag/AgCl) (0.263 V versus NHE) in 0.1 mol/L pH 6.8 PBS. Both the size and the amount of SiO2 nanoparticles could influence the electron transfer between Cyt c and the electrode. Electrostatic interaction which is between the negative nanoparticle surface and positively charged amino acid residues on the Cyt c surface is of importance for the stability and reproducibility toward the direct electron transfer of Cyt c. It is suggested that the modification of SiO2 nanoparticles proposes a novel approach to realize the direct electrochemistry of proteins.     

Electrochemical Studies of Cytochrome c on Electrodes Modified by Single-Wall Carbon Nanotubes

Single wallcarbonnanotubes (SWNTs)modifiedgoldelectrodeswerepreparedbyusingtwodifferentmethods .Theelectrochemicalbehaviorofcytochromeconthemodifiedgoldelectrodeswasinves tigated .ThefirstkindofSWNT modifiedelectrode (notedasSWNT/Auelectrode)waspreparedbytheadsorptionofcarboxyl terminatedSWNTsfromDMFdispersiononthegoldelectrode .TheoxidativelyprocessedSWNTtipswerecovalentlymodifiedbycouplingwithamines (AET)toformamidelinkage .ViaAu—Schemicalbonding ,theself assembledmonolayerofthi…     

Electrochemical Determination of Superoxide Based on Cytochrome c Immobilized on DDAB‐Modified Powder Microelectrode

Abstract

Cytochrome c was immobilized at didodecyldimethylammonium bromide (DDAB)‐modified powder microelectrode and presented quasi‐reversible electrochemistry. The apparent surface coverage of cytochrome c is greatly enhanced by using powder microelectrode technique, which is 1.21×10^?8 mol/cm², more than one to three orders of magnitude larger than that obtained with thiol and DNA‐modified Au electrode. The cytochrome c modified powder microelectrode was applied for the amperometric determination of superoxide generated by the reaction of hypoxanthine with xanthine oxidase (XOD) in the presence of dioxygen.

The detection sensitivity of the modified powder microelectrode is 0.74 µA/cm² µM, which is larger than that reported in previous publications. The detection limit of the modified powder microelectrode (PME) is 0.5 µM, and the linear detection range is 0.86～5.93 µM (values of the concentration are all in terms of hypoxanthine concentration in the solution).     

Self-assembled mono- and bilayers on gold electrodes to assess antioxidants—a comparative study

Oxidative stress is considered as an imbalance of reactive species over antioxidants, leading to diseases and cell death. Various methods have been developed to determine the antioxidant potential of natural or synthetic compounds based on the ability to scavenge free radicals. However, most of them lack biological relevance. Here, a gold-based self-assembled monolayer (SAM) was compared with a gold-supported lipid bilayer as models for the mammalian cell membrane to evaluate the free radical scavenging activity of different antioxidants. The oxidative damage induced by reactive species was verified by cyclic and differential pulse voltammetry and measured by the increase of electrochemical peak current of a redox probe. Trolox, caffeic acid (CA), epigallocatechin gallate (EGCG), ascorbic acid (AA), and ferulic acid (FA) were used as model antioxidants. The change in the decrease of the electrochemical signal reflecting oxidative membrane damage confirms the expected protective role. Both model systems showed similar efficacies of each antioxidant, the achieved order of radical scavenging potential is as follows: Trolox > CA > EGCG > AA > FA. The results showed that the electrochemical assay with SAM-modified electrodes is a stable and powerful tool to estimate qualitatively the antioxidative activity of a compound with respect to cell membrane protection against biologically relevant reactive species.

     

Direct Electrochemistry of Cytochrome c on EDTA-ZrO_2 Organic-inorganic Hybrid Film Modified Electrodes

Introduction Cytochrome c (cyt c) is probably the most thor-oughly studied redox protein. It contains one Fe(III) redox center located in a haem unit which is approxi-mately spherical shape with 3.4 nm diameter and 12384 Dalton molecular weight. On metal electrode surfaces it usually shows a short lived, transient response. Many factors can impede direct electron transfer between electrodes and cyt. c, including adsorption onto elec-trode surfaces of macromolecular species (impurities) or de…     

Surface-enhanced Raman scattering of 4,4′-bipyridine on gold nanoparticle surfaces

The adsorption structure and mechanism of 4,4′-bipyridine (BiPy) on gold nanoparticle surfaces has been investigated by means of surface-enhanced Raman scattering (SERS). The aromatic ring of BiPy appeared to assume a perpendicular orientation with respect to the gold surface from the presence of the ν(CH) band at ∼3060 cm⁻¹. The SERS intensities of several vibrational modes of BiPy on Au were found to vary as the bulk concentration. The SERS intensities for BiPy on Au could be ascribed to both the electromagnetic (EM) and charge transfer (CT) enhancement mechanism.     

Electorchemical Studies of Cytochrome c on Electodes Modified by Single—Wall Carbon Naotubes

Single-wall carbon nanotubes(SWNTs) modified gold electrodes were prepared by using two different methods.The electrochemical behavior of cytochrome c on the modified gold electrodes was investigated.The first kind of SWNT-modified electrode (noted as SWNT/Au electrode)was prepared by the adsorption of carboxylterminated SWNTs from DMF dispersion on the gold electrode.The oxidatively processed SWNT tips were covalently modified by coupling with amines (AET) to form amide linkage.Via Au-S chemical bonding,the self-assembled monolayer of thiol-unctionalized nanotubes on gold surface was fabricated so as to prepare the others SWNT-modified electrode (noted as SWNT/AET/Au electrode).It was shown from cyclic voltammetry cxperiments that cytochrome c exhibited direct electrochemical responses on the both electrodes, but only the current of controlled diffusion existed on the SWNT/Au electrode while both the currents of controlled diffusion and adsorption of cytochrome c occurred on the SWNT/AET/Au electrode.Photoelastic Modulation Infared Reflection Absorpthion Spectroscopy (PEM-IRRAS) and Quartz Crystal Microbalance (QCM) were employed to verify the adsorption of SWNTs on the gold electrodes.The results proved that SWNTs could enhance the direct electron transfer proecss between the electrodes and redox proteins.     

Electrochemical and Spectroscopic Study on the Interaction of Cytochrome c with Anionic Lipid Vesicles

The structure and the electron-transfer of cytochrome c binding on the anionic lipid vesicles wrer analyzed by electrochemical and various spectroscopic methods.It was found that upon binding to anionic lipid membrane,the formal potential of cytochrome c shifted 30 mV negtively indicating an easier redox interaction than that in its native state.This is due to the local alteration of the coordination and the heme crevice.The structural perturbation in which a molten globule-like state is formed during binding to anionic lipid vesicles is more important.This study may help to understand the mechanism of the electron-transfer reactions of cytochrome c at the mitochondrial membrane.     

Nucleic Acid–Promoted Electron Transfer to Cytochrome c

Gold electrodes were modified with short oligonucleotides in order to facilitate the electron transfer to the small redox protein cytochrome c. DNA immobilization was followed by impedance spectroscopy. The electron exchange was found to be quasi-reversible for both the protein in solution or adsorbed at the electrode surface. Variations of base sequence or structure (DNA-PNA hybrids) of the nucleic acid promoter layer did not significantly change the electron transfer rate constant which was in the range of 0.2–1×10⁻² cm/s. Cytochrome c adsorbed at low ionic strength was coupled to molecules in solution such as superoxide or laccase. Aspects of the DNA conductivity and possibilities for DNA detection are also discussed.     

Highly conducting π-conjugated molecular junctions covalently bonded to gold electrodes

We measure electronic conductance through single conjugated molecules bonded to Au metal electrodes with direct Au-C covalent bonds using the scanning tunneling microscope based break-junction technique. We start with molecules terminated with trimethyltin end groups that cleave off in situ, resulting in formation of a direct covalent σ bond between the carbon backbone and the gold metal electrodes. The molecular carbon backbone used in this study consist of a conjugated π system that has one terminal methylene group on each end, which bonds to the electrodes, achieving large electronic coupling of the electrodes to the π system. The junctions formed with the prototypical example of 1,4-dimethylenebenzene show a conductance approaching one conductance quantum (G(0) = 2e(2)/h). Junctions formed with methylene-terminated oligophenyls with two to four phenyl units show a 100-fold increase in conductance compared with junctions formed with amine-linked oligophenyls. The conduction mechanism for these longer oligophenyls is tunneling, as they exhibit an exponential dependence of conductance on oligomer length. In addition, density functional theory based calculations for the Au-xylylene-Au junction show near-resonant transmission, with a crossover to tunneling for the longer oligomers.     

Structures of Cytochrome b_5 Mutated at the Charged Surface-Residues and Their Interactions with Cytochrome c

ＩｎｔｒｏｄｕｃｔｉｏｎＭｉｃｒｏｓｏｍａｌｃｙｔｏｃｈｒｏｍｅｂ5(Ｃｙｔｂ5)ｉｓａｍｅｍｂｅｒｏｆｃｙｔｏｃｈｒｏｍｅｂ5ｆａｍｉｌｙ ,ａｎｄｉｔｓｅｒｖｅｓａｓａｎｅｌｅｃｔｒｏｎｃａｒｒｉｅｒｉｎａｓｅｒｉｅｓｏｆｅｌｅｃｔｒｏｎ ｔｒａｎｓｆｅｒｐｒｏｃｅｓｓｅｓｉｎｂｉｏｌｏｇｉｃａｌｓｙｓ ｔｅｍｓ .1 3 Ｃｙｔｂ5ｉｓａｍｅｍｂｒａｎｅｐｒｏｔｅｉｎｗｉｔｈＭｒ～ 16ｋＤａ ,ｃｏｎｓｉｓｔｉｎｇｏｆｔｗｏｄｏｍａｉｎｓ ,ｏｎｅｈｙｄｒｏｐｈｏｂｉｃｄｏｍａｉｎｗｈｉｃｈａｎｃｈｏｒｓｔｈ…     

Implication of Threonine-Based Ionic Liquids on the Structural Stability,Binding and Activity of Cytochrome c

Ionic liquids (ILs) are useful in pharmaceutical industries and biotechnology as alternative solvents or sources for protein extraction and purification, preservation of biomolecules and for regulating the catalytic activity of enzymes. However, the binding mechanism, the non-covalent forces responsible for protein-IL interactions and dynamics of proteins in IL need to be investigated in depth for the effective use of ILs as alternatives. Herein, we disclose the molecular level understanding of the structural intactness and reactivity of a model protein cytochrome c (Cyt c) in biocompatible threonine-based ILs with the help of experimental techniques such as isothermal titration calorimetry (ITC), fluorescence spectroscopy, transmission electron microscopy (TEM) as well as molecular docking. Hydrophobic and electrostatic forces are responsible for the structural and conformational integrity of Cyt c in IL. The ITC experiments revealed the Cyt c-IL binding free energies are in the range of 10–14 kJ/mol and the molecular docking studies demonstrated that ILs interact at the surfaces of Cyt c. The results look promising as the ILs used here are non-toxic and biocompatible, and thus may find potential applications in structural biology and biotechnology.     

The Electrochemistry of Cytochrome c at a Viologen-thiol Self-Assembled Monolayer

ＴｈｅＥｌｅｃｔｒｏｃｈｅｍｉｓｔｒｙｏｆＣｙｔｏｃｈｒｏｍｅｃａｔａＶｉｏｌｏｇｅｎ－ｔｈｉｏｌＳｅｌｆ－ＡｓｓｅｍｂｌｅｄＭｏｎｏｌａｙｅｒＬＩＪｉｎｇ－ｈｏｎｇ,ＣＨＥＮＧＧｕａｎｇ－ｊｉｎ,ＤＯＮＧＳｈａｏ－ｊｕｎ（ＬａｂｏｒａｔｏｒｙｏｆＥ...     

Monolayers of photosystem II on gold electrodes with enhanced sensor response—effect of porosity and protein layer arrangement

Mass transport of the bulk of the analyte to the electrode and through the bioactive layer can be significantly improved by use of the nanoelectrode array and defined arrangement of protein film. This phenomenon has been studied by (i) atomic-force microscopy, (ii) electrochemical measurements of PSII activity, and (iii) digital simulations for an oriented monolayer of histidine-tagged photosystem II (PSII) immobilized on nitrilotriacetic acid (NTA)-modified gold electrodes. The output signal of the electrochemical biosensor is controlled by (i) mass transport from the bioactive layer to electrode and (ii) mass transport between the bulk of the analyte and the electrode. Mass transport through the bioactive layer was electrochemically studied for PSII self-assembled on gold screen-printed electrodes. A densely packed monolayer of PSII has a significant shielding effect toward the diffusion of redox mediator duroquinone (DQ). Mass transport to the planar electrode surface was improved by co-immobilization of bovine-serum albumin (BSA) as spacer biomolecule in the monolayer of PSII. Correlation between the electrochemical properties and surface arrangement of the resulting protein films was clearly observable and confirmed the improved mass-transport properties of structured enzyme monolayers. On the basis of this observation, the application of a bottom-up approach for improvement of electrode performance was proposed and digitally simulated for an infinite array of electrodes ranging in diameter from 50 nm to 5 m. The nanoelectrode array, with the optimum time window selected for measurements, enables enhancement of mass transport between the bulk of the analyte and the macroelectrode by a factor of up to 50 in comparison with classical planar electrodes. Use of a time window enables minimization of crosstalk between individual electrodes in the array. The measurements require methods which suppress the double-layer capacity.     

Proline-40 is Essential to Maintaining Cytochrome b_5''''s Stability and Its Electron Transfer with Cytochrome c

ＩｎｔｒｏｄｕｃｔｉｏｎＣｙｔｏｃｈｒｏｍｅｂ5(Ｃｙｔｂ5)ｉｓａｍｅｍｂｒａｎｅ ｂｏｕｎｄｐｒｏ ｔｅｉｎ .Ｉｔｃａｎｂｅｐｒｏｔｅｏｌｙｚｅｄｔｏｙｉｅｌｄａｓｏｌｕｂｌｅ ,ｈｙｄｒｏｐｈｉｌｉｃｄｏｍａｉｎｃｏｎｔａｉｎｉｎｇａｎｏｎ ｃｏｖａｌｅｎｔｌｙｂｏｕｎｄｈｅｍｅｇｒｏｕｐ .Ｃｙｔｂ5ｉｓｉｎｖｏｌｖｅｄｉｎｅｌｅｃｔｒｏｎｔｒａｎｓｆｅｒｗｉｔｈａｖａｒｉｅｔｙｏｆｐｒｏｔｅｉｎｓ,ｓｕｃｈａｓｃｙｔｏｃｈｒｏｍｅｃ (Ｃｙｔｃ) ,1 3 ｍｅｔｍｙｏ ｇｌｏｂｉｎ ,2 ｍｅｔｈｅｍｏｇｌｏ…     

Cytochrome c–dimyristoylphosphatidylglycerol interactions studied by asymmetrical flow field-flow fractionation

Lipid membranes are well recognized ligands that bind peripheral and integral proteins in a specific manner and regulate their function. Cytochrome c (cyt c) is one of the partner peripheral protein that binds to the lipid membranes via electrostatic and hydrophobic interactions. In this study, asymmetrical flow field-flow fractionation (AsFlFFF) was used to compare the interactions of cyt c with the acidic phospholipid 1,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol (DMPG), oleic acid (OA), and sodium dodecyl sulfate (SDS). The influence of pH and the cyt c–lipid molar mass ratios were evaluated by monitoring the diffusion coefficients and particle diameter distributions obtained for the free and lipid-bound protein. The hydrodynamic particle diameter of cyt c (pI 10) was 4.1 nm at pH 11.4 and around 4.2 nm at pH 7.0 and 8.0. Standard molar mass marker proteins were used for calibration to obtain the molar masses of free cyt c and its complexes with lipids. AsFlFFF revealed the binding of cyt c to DMPG and to OA to be mainly electrostatic. In the absence of electrostatic interactions, minor complex formation occurred, possibly due to the extended lipid anchorage involving the hydrophobic cavity of cyt c and the hydrocarbon chains of DMPG or SDS. The possibility of the formation of the molten globule state of cyt c, induced by the interaction between cyt c and lipids, is discussed.Electronic Supplementary Material Supplementary material is available for this article at     

DFT Calculations for Mössbauer Properties on Dinuclear Center Models of the Resting Oxidized Cytochrome c Oxidase

Mössbauer isomer shift and quadrupole splitting properties have been calculated using the OLYP-D3(BJ) density functional method on previously obtained (W.-G. Han Du, et al., Inorg Chem. 2020 , 59, 8906–8915) geometry optimized Fe_a3³⁺−H₂O−Cu_B²⁺ dinuclear center (DNC) clusters of the resting oxidized ( O state) “as-isolated” cytochrome c oxidase (CcO). The calculated results are highly consistent with the available experimental observations. The calculations have also shown that the structural heterogeneities of the O state DNCs implicated by the Mössbauer experiments are likely consequences of various factors, particularly the variable positions of the central H₂O molecule between the Fe_a3³⁺ and Cu_B²⁺ sites in different DNCs, whether or not this central H₂O molecule has H-bonding interaction with another H₂O molecule, the different spin states having similar energies for the Fe_a3³⁺ sites, and whether the Fe_a3³⁺ and Cu_B²⁺ sites are ferromagnetically or antiferromagnetically spin-coupled.