Properties Of Cytochrome Research Articles

Electro-Infiltration of Cytochrome C into a Porous Silicon Network, and Its Effect on Nucleation and Protein Crystallization—Studies of the Electrical Properties of Porous Silicon Layer-Protein Systems for Applications in Electron-Transfer Biomolecular Devices

In this work, we report the electrical properties of cytochrome C (Cyt C) inside porous silicon (PSi). We first used two techniques of protein infiltration: classic sitting drop and electrochemical migration methods. The electrochemically assisted cell, used for the infiltration by electro-migration, improved the Cyt C nucleation and the crystallization behavior due to the PSi. We were able to carry out the crystallization thanks to the previous infiltration of proteins inside the Si pores network. We then continued the protein crystal growth through a vapor diffusion set-up. Secondly, we applied both forward and reverse bias currents only to the infiltrated Cyt C. Finally, the electrical characteristics were compared to the control (the protein molecules of which were not infiltrated) and to the samples without protein infiltration. The linker used in the sitting drop method influenced the electrical properties, which showed a modification in the current density. The simple drop method showed a current density of ~42 A/cm2; when employing the electrochemical cell technique, the current density was ~318 A/cm2; for the crystallized structures, it was ~0.908 A/cm2.

The pH Dependent Protein Structure Transitions and Related Spin-State Transition of Cytochrome c′ from Alcaligenes xylosoxidans NCIMB 11015

Abstract The unusual magnetic/spectroscopic properties of Cytochrome c′ (Cyt c′) have been discussed, especially concerning the possibility of a quantum mechanically mixed-spin configuration of heme Fe(III). Here, four unique-spin species were identified from the magnetic circular dichroism (MCD) spectra of Cyt c′ from Alcaligenes xylosoxidans (AxCyt c′). The electrospray ionization mass spectrometric (ESI-MS) and circular dichroism (CD) spectroscopic data showed the overall conformation of AxCyt c′ was unchanged, in complete contrast to the drastic changes in the heme MCD spectra over the range of pH 3.5 and 11.8. The pH dependency of ESI-MS, electronic absorption, MCD, and CD spectroscopic properties of AxCyt c′ enabled us to reveal the undiscovered correlation between the protein-folding state and the electronic structure of the active site as a function of pH. The mechanism of alkaline spin-state transition through the rearrangement of the hydrogen-bonding linkage between Helix C and D is also proposed on the basis of atomic resolution crystal structure analyses (A. Takashina, M. Unno, T. Kohzuma, Chem. Lett. 2015, 44, 268).

Electroanalytical properties of cytochrome c with direct electron transfer on graphene/gold nanoparticles chitosan modified glass carbon electrode

A nitrite (NO2−) sensor has been fabricated by immobilizing cytochrome c (Cyt c) on a graphene/gold nanoparticle-chitosan (Gra/Au NPs–Chit) modified glass carbon electrode (GCE). Graphene/gold nanoparticles (Gra/Au NPs) nanocomposite was prepared by dispersing graphene in an aqueous solution of β-cyclodextrin (β-CD) and hydrogen tetrachloroaurate and followed by NaBH4 reduction. An aliquot of Gra/Au NPs in a 0.50 wt% chitosan solution was drop-coated on the surface of a GCE to form a Gra/Au NPs–Chit/GCE which was subsequently adsorbed with Cyt c. The Cyt c–Gra/Au NPs–Chit/GCE shows a quasi-reversible cyclic voltammetric redox couple at a formal potential of −0.037 V (vs. SCE) in 0.10 M pH 7.0 phosphate buffer solution, suggesting that Gra/Au NPs–Chit film offers a biocompatible microenvironment for the Cyt c. It was found that the Cyt c–Gra/Au NPs–Chit sensor displays good electrocatalytic activity for NO2− oxidation at a relatively low working potential. The sensor possesses a linear response range of 10.0–420.0 μM NO2− with a correlation coefficient of 0.9989 and a sensitivity of 110.0 μA mM−1. The limit of detection is determined as 2.46 μM NO2− based on a signal-to-noise ratio of 3. The proposed sensor has been applied successfully to the determination of NO2− in spiked and real water samples.

Cytochromes—fascinating molecular machines

A huge number of functions and reactions can be performed by proteins merely based on their amino acids and their spatial arrangement which results in a specific geometric and electronic environment. The diversity and efficiency of functions, however, can be increased by protein-bound cofactors which fulfil their task in close interaction with the surrounding amino acid side chains. Among these cofactors, metals play an important role. They can be bound to proteins directly via specific amino acids such as cysteine and histidine, for example in zinc finger proteins or in iron sulfur cluster proteins. Another possibility is their integration into the 3D-structure of a protein via a prosthetic group. Porphyrine or porphyrine-like molecules with copper or iron as central bound metal ions constitute important structures in this respect. The complexed metal ions are involved in different functions such as mere ligand binding, electron transport or enzyme catalytic reactions. Cytochromes are obviously the most prominent proteins among this group of metallo-enzymes and fulfil important tasks within the cell such as components of the respiratory chain and the photosynthetic apparatus and also as enzymes, the cytochromes P450, which perform a huge number of different oxidation reactions with thousands of substrates. The functional diversity of cytochromes indicates that under the term ‘cytochromes’ different types of hemecontaining proteins are summarized. Besides large differences in the protein structures, a distinction can be made with respect to the coordination of the heme iron. In merely electron transferring cytochromes one ligand for the central iron is the imidazol nitrogen of a histidine residue. In contrast, the cysteine thiol group forms the corresponding ligand in cytochromes P450 and some related enzymes, therefore called heme-thiolate proteins for distinction. These structural features have an impact of the spectroscopic properties of the cytochrome types. As a functional respiratory chain is highly essential for each cell, a functional photosynthesis machine is required in plants, in addition. Probably for this reason, components of these sensitive systems are evolutionary highly conserved. It is noted that based on amino acid and DNA base sequences cytochrome c is for a long time used as a protein to trace phylogenetic and evolutionary relationships between species and higher order taxa. Cytochrome c is the smallest and simplest of the cytochromes of the respiratory chain and due to the fact that it is soluble and easy to handle, it became a model system, often studied with respect to multiple aspects covering evolutionary biology, protein structure, protein biogenesis, biophysics and physiology. These are summarized by Julie Stevens who also focuses on the post-translational maturation of the protein (DOI: 10.1039/c0mt00089b). The buildup of working electron transport chains does not only require the concerted biosynthesis and correct assembly of the involved multi-protein complexes and even supramolecular structures but also of the single holo-proteins such as cytochromes and iron sulfur cluster proteins. Correspondingly, three papers of the Themed Issue deal with specific aspects of cytochrome c biosynthesis. Achievement of the correct three-dimensional structure as well as the proper insertion and covalent attachment of the porphyrine ring system are important for the function of cytochrome c. During protein folding different intermediate stages such as molten and pre-molten globules can be distinguished. This aspect is addressed by the contribution of Ahmad et al. who also consider factors which can influence the structure and function of these states (DOI: 10.1039/c0mt00078g). The supply of heme to apo-cytochrome c is facilitated by the chaperone CcmE. Heme moieties loaded with several metals such as zinc and tin aside from iron show low or no affinity to the chaperone. In the case of Co the heme is reversibly bound to cytochrome c and not attached covalently. Ferguson et al. investigated the reasons for the different chemical behaviour of the metal–heme complexes (DOI: 10.1039/c0mt00085j). For the covalent attachment of iron loaded heme to cytochrome c specific enzymes and specific structural features are required such as the CXXCH binding motif. Alterations of the amino acid sequence of the binding motif or of adjacent areas in connection with the several involved enzyme systems give insight into the requirements for proper and efficient heme attachment (Kleingardner and Bren, DOI: 10.1039/c0mt00086h). The correct spatial positioning and orientation of the heme iron within the protein as well as the surface properties of cytochrome c play an important role as they define the interactions with the redox partners such as cytochrome aa3. Bertini et al. identified three major configuration states of cytochrome which are important for the relevant a Leibniz Research Centre for Working Environment and Human Factors (IfADo), Ardeystr. 67, 44139 Dortmund, Germany b BAM Federal Institute for Materials Research and Testing, Richard Willstaetter-Str. 11, 12489, Berlin, Germany Metallomics Dynamic Article Links

Molecular Basis of Coupled Protein and Electron Transfer Dynamics of Cytochrome c in Biomimetic Complexes

Direct electron transfer (ET) of redox proteins immobilized on biomimetic or biocompatible electrodes represents an active field of fundamental and applied research. In this context, several groups have reported for a variety of proteins unexpected distance dependencies of the ET rate, whose origin remains largely speculative and controversial, but appears to be a quite general phenomenon. Here we have employed molecular dynamics (MD) simulations and electron pathway analyses to study the ET properties of cytochrome c (Cyt) electrostatically immobilized on Au coated by carboxyl-terminated alkylthiols. The MD simulations and concomitant binding energy calculations allow identification of preferred binding configurations of the oxidized and reduced Cyt which are established via different lysine residues and, thus, correspond to different orientations and dipole moments. Calculations of the electronic coupling matrices for the various Cyt/self-assembled monolayer (SAM) complexes indicate that the thermodynamically preferred protein orientations do not coincide with the orientations of optimum coupling. These findings demonstrate that the ET of the immobilized Cyt is controlled by an interplay between protein dynamics and tunneling probabilities. Protein dynamics exerts two level of tuning on the electronic coupling via reorientation (coarse) and low amplitude thermal fluctuations (fine). Upon operating the Au support as an electrode, electric-field-dependent alignment of the protein dipole moment becomes an additional determinant for the protein dynamics and thus for the overall ET rate. The present results provide a consistent molecular description of previous (spectro)electrochemical data and allow conclusions concerning the coupling of protein dynamics and ET of Cyt in physiological complexes.

Relocation of the Distal Histidine in Cytochrome c Peroxidase: Properties of CcP(W51H), CcP(W51H/H52W), and CcP(W51H/H52L)

Many heme proteins have distal histidine residues that play important roles in determining heme protein reactivity. These distal histidines are in significantly different orientations,and distances from the heme iron in different heme proteins and the position of the distal histidine relative to the heme iron can influence reactivity at the heme center. To explore the effect of distal histidine position on the properties of cytochrome c peroxidase (CcP), three CcP mutants in which tryptophan 51 was replaced with a histidine residue were constructed. All three mutants, CcP(W51H), CcP(W51H/H52W), and CcP(W51H/H52L), have altered electronic absorption spectra, indicating that the heme group in the mutants is six-coordinate rather than five-coordinate as it is in wild-type CcP. The hydrogen peroxide reaction rate is 56-6200-fold slower for the mutants than for wild-type CcP. All three mutants form a CcP Compound I-like intermediate, in which the Fe(IV) site decays between 500 and 3000 times more rapidly than the Fe(IV) site in wild-type CcP Compound I. The W51H mutations have a weaker effect on cyanide binding, with the cyanide affinity only 2-8 times weaker than for CcP. The cyanide association rate constants are between 5 and 85 times slower for the W51H mutants, while the cyanide dissociation rate constants range from 3 times slower to 6 times faster than those of wild-type CcP.

Engineering, expression, purification, and physicochemical characterization of a chimeric protein, full-length cytochrome b 5-green fluorescence protein (HMWb5-EGFP)

In this article we report on construction of expression vector, heterologous expression in Escherichia coli, isolation, purification, and physicochemical characterization of an artificial chimeric protein HMWb(5)-EGFP consisting of full-length cytochrome b(5) (HMWb(5)) and green fluorescence protein (EGFP) from Aequorea. Optimization of expression conditions yielded an expression level up to 1500 nmol of chimeric protein per liter of culture. Recombinant chimeric protein HMWb(5)-EGFP was purified from cell membranes by using metal-affinity chromatography. It possesses physicochemical, spectral, and fluorescence properties of cytochrome b(5) and EGFP indicating independent character of protein folding in frames of the chimera. It is shown that there is a fluorescent resonance energy transfer in HMWb(5)-EGFP between the fluorophore of EGFP and heme of cytochrome b(5), and the distance between chromophores in the chimeric protein is approximately 67.3 A. The chimeric protein was shown to exist as a monomer in aqueous solution in the presence of detergents. The data indicate that the HMWb(5)-EGFP designed in the present work is a very promising model for modern biosensors and an instrument to study protein-protein interactions.

Orientations of axially coordinated imidazoles and pyridines in crystal structures of model systems of cytochromes

Many properties of cytochromes and model systems depend on orientations of axial ligands. In this work, we elucidated the role of porphyrin substituents on orientation of axial ligands in model systems of cytochromes. The orientations of axially coordinated imidazoles and pyridines in crystal structures of model systems of cytochromes were analyzed and data were compared with previous quantum-chemical calculations. The results show that eight ethyl groups on porphyrin ring strongly favor parallel orientation, hence, in all these complexes axial ligands, pyridines or imidazoles, are mutually parallel. Four phenyl or mesityl groups at meso-carbons also favor parallel orientation but less strongly. Hence, in most of the bis-imidazole complexes the orientation is parallel, while in bis-pyridine complexes the orientation of pyridines depends on oxidation state of Fe. In bis-pyridine Fe(II) complexes orientation is parallel, in Fe(III) it is orthogonal. This analysis is in agreement with previous quantum-chemical calculations.

STM investigation of electron transport features in cytochrome c Langmuir–Blodgett films

Abstract The morphological and electron-transfer properties of cytochrome c monolayer Langmuir–Blodgett (LB) films deposited on a graphite electrode have been studied using scanning tunneling microscopy (STM) and tunneling spectroscopy techniques in a double tunnel junction configuration STM tip–cytochrome c monolayer–conducting substrate. Monolayer cytochrome c LB films were deposited successfully by horizontal lifting method via protein Langmuir monolayer formation using preliminary solubilization of the protein in water bubbles of reverse microemulsion of Aerosol OT in hexane. Electronic spectroscopy of a single cytochrome c molecule was carried out with sub-nanometer spatial resolution at the ambient conditions. The STM spectroscopic study, by recording tunneling current–bias voltage (I–V) curves, revealed nonsymmetric I–V curves with steps of variable widths and heights dependent on the STM tip position over the protein molecule. Complex-rich structure of the tunneling current–voltage spectra can be a result of the interplay between Coulomb charging effects and tunneling through discrete electronic levels of the cytochrome c molecule. It can also be determined by the tunnel junctions parameters, dependent on the state of cytochrome c molecules in the gap between the STM tip and the substrate, presence of absorbates and ‘electron paths’ effects in the protein molecule.

Catalytic and spectroscopic properties of cytochrome-c, horseradish peroxidase, and ascorbate oxidase embedded in a sol-gel silica matrix as a function of gelation time.

In this study, we investigated the optical features of the redox metal-dependent proteins cytochrome-c, horseradish peroxidase (HRP), and ascorbate oxidase embedded in a sol-gel-processed silica matrix as a function of gelation time. Circular dichroism, absorbance, and fluorescence spectroscopies revealed that the sol-gel process affects the complex structure of the dimeric ascorbate oxidase (although the prosthetic coppers still remain bound to the enzyme) but not that of monomeric cytochrome-c and HRP. Any modifications in ascorbate oxidase occurred in the initial gelation phase; the drying process induced no further alterations and the enzyme remained stable for months. Unfolding-refolding experiments on cytochrome-c revealed severely restricted motility in the protein moiety in the xerogel, the concentrated matrix that forms after drying. The diffusion time of the solvent within the matrix, which regulated the enzyme-substrate reaction rate, depended on the thickness of the monolith, not on the dryness of the specimen.

Peroxidative damage to cardiac mitochondria: cytochrome oxidase and cardiolipin alterations

Rat heart mitochondrial membranes exposed to the free radicals generating system tert-butylhydroperoxide/Cu 2+ undergo lipid peroxidation as evidenced by the accumulation of thyobarbituric acid reactive substances. Mitochondrial lipid peroxidation resulted in a marked loss of both cytochrome c oxidase activity and cardiolipin content. The alterations in the properties of cytochrome c oxidase were confined to a decrease in the maximal activity ( V max) with no change in the affinity ( K m) with respect to the substrate cytochrome c. Various lipid soluble antioxidants could prevent the lipid peroxidation reaction and the associated loss of cytochrome c oxidase activity. External added cardiolipin but no other phospholipids, nor peroxidized cardiolipin was able to prevent the loss of cytochrome oxidase activity induced by lipid peroxidation. These results establish a close correlation between oxidative damage to cardiolipin and alterations in the cytochrome oxidase activity and may prove useful in probing molecular mechanism of free radicals induced peroxidative damage of mitochondria which has been proposed to contribute to aging and to chronic degenerative diseases.

Electrostatic properties of cytochrome c variants and of the cytochrome c-cytochrome b5 complex.

Electrostatic properties of cytochrome c variants and of the cytochrome c-cytochrome b5 complex.

Vibrational structure of the formyl group on heme a. Implications on the properties of cytochrome c oxidase

Resonance Raman spectra have been recorded for heme a derivatives in which the oxygen atom of the formyl group has been isotopically labeled and for Schiff base derivatives of heme a in which the Schiff base nitrogen has been isotopically labeled. The 14N-15N isotope shift in the C = N stretching mode of the Schiff base is close to the theoretically predicted shift for an isolated C = N group for both the ferric and ferrous oxidation states and in both aqueous and nonaqueous solutions. In contrast, the 16O-18O isotope shift of the C = O stretching mode of the formyl group is significantly smaller than that predicted for an isolated C = O group and is also dependent on whether the environment is aqueous or nonaqueous. This differences between the theoretically predicted shifts and the observed shifts are attributed to coupling of the C = O stretching mode to as yet unidentified modes of the heme. The complex behavior of the C = O stretching vibration precludes the possibility of making simple interpretations of frequency shifts of this mode in cytochrome c oxidase.

Partial purification of sulfite oxidase from Thiobacillus neapolitanus

Sulfite oxidase was purified 172-fold from cell extract of Thiobacillus neapolitanus (protein A). The enzymatic activity was stimulated by protein B which shows properties of cytochrome c and flavoprotein. Sulfite oxidase was inhibited 20% by the addition of AMP.

Purification and properties of cytochrome P-450-dependent 14 α-sterol demethylase from Candida albicans

The purification of cytochrome P-450-dependent 14 alpha-sterol demethylase (P-450DM) from the important opportunistic fungal pathogen, Candida albicans, is described. Optimal purification (875-fold) was achieved by extracting the cytochrome from microsomes with sodium cholate followed by hydroxyapatite, octyl-Sepharose and CM-Sepharose chromatographies, giving a cytochrome preparation of 17.5 nmol/mg of protein. By the use of SDS/polyacrylamide-gel electrophoresis the cytochrome was judged to be highly purified on the basis of Coomassie Brilliant Blue staining of protein. The Mr of P-450DM was estimated to be 51,000. The absorption spectrum of oxidized P-450DM was characteristic of a low-spin cytochrome, and its reduced CO complex had a Soret absorption peak at 447 nm. When reconstituted in a model membrane system of dilauroylphosphatidylcholine with NADPH and O2, P-450DM catalysed the complete 14 alpha-demethylation of lanosterol, which was inhibited by CO. The cytochrome appeared to have a high degree of substrate specificity; it was unable to oxidize a number of xenobiotic compounds in the reconstituted assay.

Isolation and Comparison of Molecular Properties of Cytochrome £-559 from Both Spinach Thylakoids and PS II Particles

Abstract Cytochrome b-559. Spinach Thylakoids. PS II Particles. High and Low Potential. Interconvertible Forms Cytochrome b-559 has been purified from both spinach (Spinacia oleracea L.) thylakoids and photosystem II particles in order to compare some of its more polemic molecular properties. In the two cases, cytochrome 6-559 (which is initially present in its reduced high-potential form and in its oxidized low-potential form) is purified as a single species by preparative disc electrophoresis in Triton-containing polyacrylamide gel, the isolated protein exhibiting similar molecular mass and almost identical low midpoint redox potential and absorption spectra. Moreover, sodium dodecyl sulfate polyacrylamide gel electrophoresis of the purified cytochrom e b-559 reveals, again in both cases, only one polypeptide band of similar molecular mass. Integration o f the isolated low-potential form into liposomes partly restores the high-potential form. These data corroborate our previous results and indicate that there exists in thylakoids only a cytochrome b -559 molecular species tightly bound to PS II and that the two naturally occurring low-and high-potential couples are physiological and correspond to interconvertible states of the genuine hem e protein.

Catalytic properties of cytochrome c oxidase purified from Nitrosomonas europaea.

Cytochrome c oxidase of Nitrosomonas europaea reacts with not only the native cytochrome c (N. europaea cytochrome c-552) but also horse and yeast cytochromes c. The effects on its reactivity of various reagents were very different between the reactions with the native and eukaryotic cytochromes c as the electron donors. The oxidation of eukaryotic ferrocytochrome c by the oxidase was activated by addition of anionic detergents such as sodium dodecyl sulfate and sodium cholate, and anionic phospholipids such as cardiolipin, phosphatidylserine, phosphatidylinositol, and phosphatidylethanolamine, while the reaction was not activated by Triton X-100, Tween 20, or phosphatidylcholine. However, the reaction with the native cytochrome c of the enzyme was hardly affected by any of the detergents and phospholipids mentioned above, while it was activated by the presence of poly-L-lysine.

Resolution of the circular dichroism spectra of the mitochondrial cytochrome bc1 complex

The circular dichroic spectrum of the mitochondrial cytochrome bc 1 complex isolated from bovine heart has been resolved into the contributions from the prosthetic groups: cytochrome c 1, the ‘Rieske’ iron-sulphur centre and the two b cytochromes. It is apparent that firstly, the circular dichroism (CD) properties of cytochrome c 1 within the bc 1 complex differ from those found in the isolated cytochrome c 1 and secondly, both the oxidized and reduced b cytochromes exhibit an intense spectrum of bilobic shape, with the wavelengths of the cross-over points closely corresponding to those of the maxima in the optical absorbance spectra. These latter CD features are discussed in relation to the proposed structure of cytochrome b.

Properties of cytochrome c and methanol dehydrogenase produced by wild type and mutant strains of Methylomonas sp. in continuous culture

The cytochrome c production of the wild type strain and a mutant strain, YK 56, of Methylomonas sp. grown with excess methanol was higher than wild with limited methanol. The wild type strain grown under both conditions contained two soluble cytochromes c ( c-I and c-II), though the mutant strain contained three ( c-I, c-II, and c-III). The proportions of cytochromes c-II and c-III of the mutant strain damage changed according to the culture conditions. The methanol dehydrogenase of the wild type and mutant strains was purified and characterized. The enzymes were similar; they consisted of two subunits and their molecular weight was 120,000. The reactivity of cytochromes c with methanol dehydrogenase was investigated.

Purification and some properties of cytochrome c' from a strain of Achromobacter xylosoxidans.

Cytochrome c' was crystallized from Achromobacter xylosoxidans GIFU 543. The cytochrome was a basic protein and its molecular weight was 28,000. The pyridine ferrohemochrome showed absorption peaks at 415, 521, and 551 nm. The absorption spectra of the oxidized and reduced forms at neutral pH were almost the same as those of other cytochromes c' reported already. The reduced cytochrome c' reacted with CO and NO, and the NO complex showed a characteristic absorption spectrum. The midpoint redox potential of the hemoprotein was measured to be + 110 mV at pH 7.2.