Redox Properties Of Cytochrome Research Articles

Redox heme-proteins mediated fluorescence of CdSe/ZnS quantum dots

The redox properties of cytochrome c (Cyt c), hemoglobin (Hb) and myoglobin (Mb) were studied based on electrostatic interactions between Thioglycolic acid (TGA) capped CdSe/ZnS quantum dots (QDs) and proteins. Results indicated that only Cyt c quenched the fluorescence of the QDs at pH>8.0. Under the optimized conditions, a significant fluorescence recovery of the QDs’ system was observed when the reduced form of Cyt c incubated with TGA capped QDs, however, the reduced state of Hb and Mb resulted in a more fluorescence quenching on the same size of QDs. Interestingly, the fluorescence changes of QDs-proteins could be switched by modulating the redox potentials of proteins-attached QDs. Moreover, only the oxidized Cyt c form was reduced by the generated O2- that significantly enhanced the fluorescence of the QDs’ system, which was also demonstrated by fluorescence imaging in HeLa cells.

Phosphate mediated adsorption and electron transfer of cytochrome c. A time-resolved SERR spectroelectrochemical study

The study of proteins immobilized on biomimetic or biocompatible electrodes represents an active field of research as it pursues both fundamental and technological interests. In this context, adsorption and redox properties of cytochrome c (Cyt) on different electrode surfaces have been extensively reported, although in some cases with contradictory results. Here we report a SERR spectroelectrochemical study of the adsorption and electron transfer behaviour of the basic protein Cyt on electrodes coated with amino-terminated monolayers. The obtained results show that inorganic phosphate (Pi) and ATP anions are able to mediate high affinity binding of the protein with preservation of the native structure and rendering an average orientation that guarantees efficient pathways for direct electron transfer. These findings aid the design of Cyt-based bioelectronic devices and understanding the modulation by Pi and ATP of physiological functions of Cyt.

PH-Induced Changes in Adsorbed Cytochrome c. Voltammetric and Surface-Enhanced Resonance Raman Characterization Performed Simultaneously at Chemically Modified Silver Electrodes

The influence of pH on the redox properties of cytochrome c (cyt c) adsorbed on roughened silver electrodes chemically modified with a self-assembled monolayer (SAM) of 11-mercapto-1-undecanoic acid (MUA) was studied with voltammetric techniques in combination with surface-enhanced resonance Raman scattering (SERRS). The experiments were performed simultaneously on the same electrode sample in a homemade spectroelectrochemical cell suitable for such applications. At pH 7.0 cyt c was found in its native state; at higher pH values (ranging from 8.0 to 9.0) the redox properties of the adsorbed protein varied considerably, featuring a redox behavior which does not resemble the one reported for the alkaline transition. Our results instead indicate the presence of an electrochemically inactive 6cLS species immobilized on MUA at pH 9.0. The pH-induced conformational changes observed for cyt c immobilized on the SAM of MUA were found to be repeatable and chemically reversible, meaning that the recovery of the electrochemical signal due to the native protein occurred instantaneously (on the second time scale) when the electrode was switched back to pH 7.0. The pH-induced changes observed were attributed to a conformational change involving a heme reorientation with respect to the electrode surface.

Evidence for a Novel Quinone-Binding Site in the Photosystem II (PS II) Complex That Regulates the Redox Potential of Cytochrome b559

The present study provides a thorough analysis of effects on the redox properties of cytochrome (Cyt) b559 induced by two photosystem II (PS II) herbicides [3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,4-dinitro-6-sec-butylphenol (dinoseb)], an acceleration of the deactivation reactions of system Y (ADRY) agent carbonylcyanide-m-chlorophenylhydrazone (CCCP), and the lipophilic PS II electron-donor tetraphenylboron (TPB) in PS II membrane fragments from higher plants. The obtained results revealed that (1) all four compounds selectively affected the midpoint potential (E(m)) of the high potential (HP) form of Cyt b559 without any measurable changes of the E(m) values of the intermediate potential (IP) and low potential (LP) forms; (2) the control values from +390 to +400 mV for HP Cyt b559 gradually decreased with increasing concentrations of DCMU, dinoseb, CCCP, and TPB; (3) in the presence of high TPB concentrations, a saturation of the E(m) decrease was obtained at a level of about +240 mV, whereas no saturation was observed for the other compounds at the highest concentrations used in this study; (4) the effect of the phenolic herbicide dinoseb on the E(m) is independent of the occupancy of the Q(B)-binding site by DCMU; (5) at high concentrations of TPB or dinoseb, an additional slow and irreversible transformation of HP Cyt b559 into IP Cyt b559 or a mixture of the IP and LP Cyt b559 is observed; and (6) the compounds stimulate autoxidation of HP Cyt b559 under aerobic conditions. These findings lead to the conclusion that a binding site Q(C) exists for the studied substances that is close to Cyt b559 and different from the Q(B) site. On the basis of the results of the present study and former experiments on the effect of PQ extraction and reconstitution on HP Cyt b559 [Cox, R. P., and Bendall, D. S. (1974) The functions of plastoquinone and beta-carotene in photosystem II of chloroplasts, Biochim. Biophys. Acta 347, 49-59], it is postulated that the binding of a plastoquinone (PQ) molecule to Q(C) is crucial for establishing the HP form of Cyt b559. On the other hand, the binding of plastoquinol (PQH2) to Q(C) is assumed to cause a marked decrease of E(m), thus, giving rise to a PQH2 oxidase function of Cyt b559. The possible physiological role of the Q(C) site as a regulator of the reactivity of Cyt b559 is discussed.

Redox Properties of CytochromecAdsorbed on Self-Assembled Monolayers: A Probe for Protein Conformation and Orientation

The redox behavior of cytochrome c (cyt c) adsorbed to gold electrodes modified with self-assembled monolayers (SAMs) depends on the SAM. This paper examines SAMs generated from alkanethiols terminating in trimethylammonium (1), sulfonate (2), methyl (3), amine (4), and carboxylic acid (5) groups and from an aromatic thiol (6). The redox potentials of cyt c adsorbed on SAMs of 1 and 5 are relatively close to the formal potential of native cyt c measured in solution. The redox potentials of cyt c adsorbed on SAMs of 3, 4, and 6 are significantly shifted from the formal potential, and a reduction peak at about 0.5 V more negative than the formal potential (that is, a value corresponding to a more difficult reduction) was observed in all three cases. These observations suggest that cyt c changes its conformation significantly on adsorption on these surfaces. No redox peaks were observed for cyt c adsorbed on SAMs of 2, although surface plasmon resonance (SPR) studies indicate that the SAMs of 2 irreversibly adsorbed approximately a double layer of cyt c. Mixed SAMs were also studied. Most interestingly, cyt c adsorbed on mixed SAMs formed from the combinations of 1 and 2 exhibited significantly slower electron transfer (0.3−1.2 s-1) than cyt c adsorbed on a homogeneous SAM of 1 (45 s-1). These observations suggest changes in protein orientation due to the presence of the sulfonate groups at the interface. This study suggests that electrochemical measurement can be a useful probe for the conformation and orientation of protein adsorbed on surfaces.

Redox properties of cytochrome c.

The redox properties of cytochromes (cyt) c, a ubiquitous class of heme-containing electron transport proteins, have been extensively investigated over the last two decades. The reduction potential (E degrees') is central to the chemistry of cyt c for two main reasons. First, E degrees' influences both the thermodynamic and kinetic aspects of the electron exchange reaction with redox partners. Second, this thermodynamic parameter is remarkably sensitive to changes in the properties of the heme and the protein matrix, and hence can be profitably used for the investigation of the solution chemistry of cyt c. This research area owes much to the exploitation of voltammetric techniques for the determination of E degrees' for metalloproteins, which dates back to the late 1970s. Since then, much effort has been devoted to the comprehension of the molecular factors that control E degrees' in cyt c, which include first coordination sphere effects on the heme iron, the interactions of the heme group with the surrounding polypeptide chain and the solvent, and also include medium effects related to the nature and ionic composition of the solvent, pH, the presence of potential protein ligands, and the temperature. This article provides an overview of the most significant advances made in this field recently.

1P011硫酸還元菌チトクロムc_3におけるヘムの酸化還元と動的構造

1P011硫酸還元菌チトクロムc_3におけるヘムの酸化還元と動的構造

Effects of solvent on the redox properties of cytochrome c: cyclic voltammetry and 1H NMR experiments in mixed water-dimethylsulfoxide solutions

Bovine heart cytochrome c (cyt c) was studied through cyclic voltammetry, 1H NMR and circular dichroism measurements in mixed water-dimethylsulfoxide (DMSO) solutions containing up to 50% DMSO by volume, under different conditions of temperature and pH. The effect of DMSO on the reduction potential of native cyt c was found to be determined mainly the he decrease in dielectric constant of the medium. No appreciable specific DMSO-protein interactions were detected. Instead, DMSO affects to some extent the conformation of alkaline cyt c and notably, stabilizes both redox states of this form to the detriment of the native form. In particular, DMSO lowers the p K a of the native to alkaline transition for oxidized cyt c and increases the electrochemical reversibility of the voltammetric wave of the alkaline form. DMSO-induced changes in the reduction entropy for native and alkaline cyt c were also determined and interpreted tentatively in terms of solvation properties of the heme and structural features of the protein environment.

Site-directed replacement of the invariant lysine 73 of Saccharomyces cerevisiae iso-1-cytochrome c with all ribosomally encoded amino acids

Abstract Site-directed mutagenesis has been used to replace the evolutionally invariant lysine 73 of Saccharomyces cerevisiae iso-1-cytochrome c with all 19 ribosomally encoded amino acids. This residue is implicated in the protein-protein complexation and redox properties of cytochrome c . Functional studies were performed in the context of both a multicopy and a centromeric phagemid using a yeast strain that is incapable of iso-1-cytochrome c production. Functionality was ascertained by growth on the non-fermentable carbon sources lactate and glycerol at 30°C and 37°C. Doubling times derived from growth curves in liquid glycerol media were used to quantify differences in the functionality of mutants relative to the wild type protein. The cysteine 73 mutant shows a dramatically reduced growth rate. Growth rates for transformants carrying the other mutations are no more than 50% slower than transformants carrying the wild type allele. The data suggest that thermostability is related to evolutionary conservation but that the requirement for a lysine residue at position 73 is met readily by a variety of amino acids. Therefore, the redox properties of cytochrome c and its complexation with its redox partners are not absolutely dependent on a lysine at position 73.

The redox properties of cytochromes b imposed by the membrane electrostatic environment

The effect of the dipole potential field of extended membrane spanning alpha-helices on the redox potentials of b cytochromes in energy transducing membranes has been calculated in the context of a three phase model for the membrane. In this model, the membrane contains three dielectric layers; (i) a 40-A hydrophobic membrane bilayer, with dielectric constant em = 3-4, (ii) 10-20-A interfacial layers of intermediate polarity, ein = 12-20, that consist of lipid polar head groups and peripheral protein segments, and (iii) an external infinite water medium, ew = 80. The unusually positive midpoint potential, Em = +0.4 V, of the "high potential" cytochrome b-559 of oxygenic photosynthetic membranes, a previously enigmatic property of this cytochrome, can be explained by (i) the position of the heme in the positive dipole potential region near the NH2 termini of the two parallel helices that provide its histidine ligands, and (ii) the loss of solvation energy of the heme ion due to the low dielectric constant of its surroundings, leading to an estimate of +0.31 to +0.37 V for the cytochrome Em. The known tendency of this cytochrome to undergo a large -delta Em shift upon exposure of thylakoid membranes to proteases or damaging treatments is explained by disruption of the intermediate polarity (ein) surface dielectric layer and the resulting contact of the heme with the external water medium. Application of this model to the two hemes (bn and bp) of cytochrome b of the cytochrome bc1 complex, with the two hemes placed symmetrically in the low dielectric (em) membrane bilayer, results in Em values of hemes bn and bp that are, respectively, somewhat too negative (approximately -0.1 V), and much too positive (approximately +0.3 V), leading to a potential difference, Em(bp) - Em(bn), with the wrong sign and magnitude, +0.25 V instead of -0.10 to -0.15 V. The heme potentials can only be approximately reconciled with experiment, if it is assumed that the two hemes are in different dielectric environments, with that of heme bp being more polar.

Binuclear transition-metal complexes as new reagents for selective cross-linking of proteins. Coordination of cytochrome c to dirhodium(II) .mu.-tetraacetate

This study introduces binuclear transition-metal complexes as reagents for selective covalent cross-linking of proteins. Incubation of horse cytochrome c (designated cyt) with Rh{sub 2}(OAc){sub 4} under mild conditions yields the diprotein complex, Rh{sub 2}(OAc){sub 4}(cyt){sub 2}, whose composition is established by size-exclusion chromatography, uv-vis spectroscopy, and {sup 1}H NMR spectroscopy. The protein molecules are coordinated to the Rh atoms via the imidazole (Im) rings of their His 33 residues, as shown by uv difference and {sup 1}H NMR spectroscopy, by the pH effect on the complex formation, and by the control experiments with tuna cytochrome c. The diprotein complex is stable under ordinary conditions, and yet it can be cleaved, and the native protein recovered, by treatment with a suitable strong nucleophile. Spectroscopic and electrochemical measurements show that the structural and redox properties of cytochrome c are not perturbed significantly by cross-linking. Comparison between Rh{sub 2}(OAc){sub 4}(Im){sub 2} and Rh{sub 2}(OAc){sub 4}(cyt){sub 2} shows that the complex containing small ligands is not an entirely realistic model of the complex containing proteins. In particular, the enhanced stability of the latter toward hydrolysis may be due to steric bulk of the protein ligands and to hydrogen bonds that amino acid side chains more » may form with the inorganic link. Some of the findings of this study may pertain to the mechanism of antitumor action of the Rh{sub 2}(RCOO){sub 4} complexes. 86 refs., 2 tabs. « less

Modification of the structural and redox properties of cytochrome c by heteropolytungstate binding

Compex formation between horse heart ferricytochrome c and large three-dimensional polyanions has been investigated, in order to study the influence of surface electrostatic interactions on the structural and redox properties of cytochrome c. Cytochrome c binds the large heteropolytungstates (NaSh 9W 21O 86) 18− and (KAs 4W 40O 140) 27− with a 1/1 polyanion/cytochrome c ratio, and the smaller ion (SiW 11O 39) 8− with a 2/1 ratio. Upon complexation, cytochrome c undergoes structural changes that are dependent on the size and charge of the polyanion, and on the pH and ionic strength of the medium. Three different forms of complexed cytochrome c have been characterized by optical and EPR spectroscopies, in the pH range 6.5–8: an N form, close to the native structure, an A form, analogous to cytochrome c in acidic medium, and a novel B form in which the heme pocket is open but the iron remains low-spin. The redox potential of cytochrome c is lowered to 250–220 mV (vs. NHE) in the N form, and to 80 mV in the B form.

Correlations studies between structural and redox properties of cytochromes C 3

Individual redox potential values are determined for different cytochromes c 3. These polyhaemic cytochromes can be divided into two groups: one characterized by only one marked reduction step, the other one giving at least two well-marked reduction steps corresponding to redox potential values ranging from −0.120 to −0.400 V. Correlations between potential values and structural data are discussed.

Comparative studies of monohemic bacterial C-type cytochromes. Redox and optical properties of [formula omitted] Norway cytochrome c 553(550) and [formula omitted] cytochrome c 551

Redox properties of cytochrome c 553(550) from Desulfovibrio desulfuricans Norway (E′ o = 0.04 ± 0.02 V/NHE) and cytochrome c 551 from P. aeruginosa (E′ o = 0.25 ± 0.02 V/NHE) are compared with those of some monohemic c-type cytochromes. The pK value for the equilibrium between the pH-dependent forms of cytochrome c 553(550) (pK = 10.3 ± 0.1) has been also determined. It is to be noted that the difference between redox potentials can extend to nearly 250 mV, though the axial heme ligands are identical. Structural reasons have to be invoked to explain these variations.

The effect of complex-formation with polyanions on the redox properties of cytochrome c.

1. The stable complex formed between mammalian cytochrome c and phosvitin at low ionic strength was studied by partition in an aqueous two-phase system. Oxidized cytochrome c binds to phosvitin with a higher affinity than reduced cytochrome c. The difference was equivalent to a decrease of the redox potential by 22 mV on binding. 2. Complex-formation with phosvitin strongly inhibited the reaction of cytochrome c with reagents that react as negatively charged species, such as ascorbate, dithionite, ferricyanide and tetrachlorobenzoquinol. Reaction with uncharged reagents such as NNN'N'-tetramethylphenylenediamine and the reduced form of the N-methylphenazonium ion (present as the methylsulphate) was little affected by complex-formation, whereas oxidation of the reduced cytochrome by the positively charged tris-(phenanthroline)cobalt(III) ion was greatly stimulated. 3. A similar pattern of inhibition and stimulation of reaction rates was observed when phosvitin was replaced by other macromolecular polyanions such as dextran sulphate and heparin, indicating that the results were a general property of complex-formation with polyanions. A weaker but qualitatively similar effect was observed on addition of inositol hexaphosphate and ATP. 4. It is suggested that the effects of complex-formation with polyanions on the reactivity of cytochrome c with redox reagents are mainly the result of replacing the positive charge on the free cytochrome by a net negative charge. Any steric effects on polyanion binding are small in comparison with such electrostatic effects.

The influence of pH on the EPR and redox properties of cytochrome c oxidase in detergent solution and in phospholipid vesicles

The EPR signals of oxidized and partially reduced cytochrome oxidase have been studied at pH 6.4, 7.4, and 8.4. Isolated cytochrome oxidase in both non-ionic detergent solution and in phospholipid vesicles has been used in reductive titrations with ferrocytochrome c. The g values of the low- and high-field parts of the low-spin heme signal in oxidized cytochrome oxidase are shown to be pH dependent. In reductive titrations, low-spin heme signals at g 2.6 as well as rhombic and nearly axial high-spin heme signals are found at pH 8.4, while the only heme signals appearing at pH 6.4 are two nearly axial g 6 signals. This pH dependence is shifted in the vesicles. The g 2.6 signals formed in titrations with ferrocytochrome c at pH 8.4 correspond maximally to 0.25–0.35 heme per functional unit ( aa 3) of cytochrome oxidase in detergent solution and to 0.22 heme in vesicle oxidase. The total amount of high-spin heme signals at g 6 found in partially reduced enzyme is 0.45–0.6 at pH 6.4 and 0.1–0.2 at pH 8.4. In titrations of cytochrome oxidase in detergent solution the g 1.45 and g 2 signals disappear with fewer equivalents of ferrocytochrome c added at pH 8.4 compared to pH 6.4. The results indicate that the environment of the hemes varies with the pH. One change is interpreted as cytochrome a 3 being converted from a high-spin to a low-spin form when the pH is increased. Possibly this transition is related to a change of a liganded H 2O to OH − with a concomitant decrease of the redox potential. Oxidase in phosphatidylcholine vesicles is found to behave as if it experiences a pH, one unit lower than that of the medium.