Photoreduction Of Cytochrome Research Articles

Spectroscopic insights into the Photoreduction of Cytochrome c with UVA-Vis Light Irradiation

With the particular conjugation structure in the heme prosthetic group, Cyt c shows unusual functions similar to chlorophyll while irradiated by specific wavelength of UV-Vis lights. To further reveal mechanism of the photo-irradiation of Cyt c, we then studied various external factors that may influence the photo induced process. The absorbance intensity increase of band (317 nm) and Q band (520 nm and 549 nm)indicated Cyt c in phosphate-buffered saline within N2 atmosphere was photoreduced to Fe(II) Cyt c. Irradiated by 410 nm, the photoreduction process was facilitated by Met. But Trp, Tyr and Phe impeded the process due to their light absorbance abilities. In addition, the results of fluorescence and CD spectra indicated that the microenvironment polarity of Trp residue varied during the photoreduction process. And the secondary structure of Cyt c changed with lower α-helix/βsheet ratio. The photoreduction mechanism of Cyt c was intramolecular electron transfer and porphyrin cation radicals were generated. The protein structure of Cyt c changed as well as part of the photoreduction.

Spectral Study on the Photoreduction of Cytochrome c under Macromolecular Crowding

The ferric Cyt c can be photoreduced to the ferrous state by photoexcitation in dilute solution. However, these researches neglected the high crowded conditions in cell environment. In this work, we applied UV-Vis absorption, synchro- nous fluorescence and CD spectra to study the photoreduction process of Cyt c and its dependence on the external environ- ment under macromolecular crowded conditions. The UV-Vis absorption spectrum data showed that with addition of Dex- tran70 or Ficoll70 the hydrophobic microenvironment of heme in Cyt c was changed and the photoreduction process of Cyt c was facilitated. In addition, the photoreduction extent was much larger in Ficoll70 solution than that in Dextran70. Under macromolecular crowded conditions, we used four wavelengths of light to irradiate Cyt c samples and found that the 280 nm light was favored for the reduction. The optimum temperatures of photoreduction process were 37 ℃ and 25 ℃ in the pres- ence of Dextran70 and Ficoll70, respectively. The concentration of macromolecular crowding reagents had massive influence on the photoreduction process. Photoreduction extent showed an increase with rising the concentration of Dextran70 and the optimum concentration of Ficoll70 for the photoreduction was 100 gL -1 . The photoreduction process was promoted by add- ing external amino acids and the relative rates of photoreduction with amino acids followed the order Phe>Tyr>Trp>Met. Synchronous fluorescence spectra data suggested that Dextran70 and Ficoll70 were able to emit fluorescence which was ab- sorbed by porphyrin. The CD spectra results of Cyt c after irradiation illustrated that the secondary structure of protein varied with α-helix decreased, and β-sheet increased in dilute solution, but remained unchanged after irradiation under macromo- lecular crowding solutions. Based on the data, macromolecular crowded conditions could maintain a stable structure of Cyt c and promote its photoinduced electronic transfer and reduction process. Keywords macromolecular crowding environment; photoreduction; Cytochrome c; spectroscopy; secondary structure

Photo-induced electron transfer in supramolecular materials of titania nanostructures and cytochrome c

In the present paper, we report on the molecular interaction and photochemistry of TiO2 nanoparticles (NPs) and cytochrome c systems for understanding the effects of supramolecular organization and electron transfer by using two TiO2 structures: P25 TiO2 NPs and titanate nanotubes. The adsorption and reduction of cytochrome c heme iron promoted by photo-excited TiO2, arranged as P25 TiO2 NPs and as nanotubes, were characterized using electronic absorption spectroscopy, thermogravimetric analysis, and atomic force microscopy. In an aqueous buffered suspension (pH 8.0), the mass of cytochrome c adsorbed on the P25 TiO2 NP surface was 2.3 fold lower (0.75 μg m−2) than that adsorbed on the titanate nanotubes (1.75 μg m−2). Probably due to the high coverage of titanate nanotubes by adsorbed cytochrome c, the low amount of soluble remaining protein was not as efficiently photo-reduced by this nanostructure as it was by the P25 TiO2 NPs. Cytochrome c, which desorbed from both titanium materials, did not exhibit changes in its redox properties. In the presence of the TiO2 NPs, the photo-induced electron transfer from water to soluble cytochrome c heme iron was corroborated by the following findings: (i) identification by EPR of the hydroxyl radical production during the irradiation of an aqueous suspension of TiO2 NPs, (ii) impairment of a cytochrome c reduction by photo-excited TiO2 in the presence of dioxane, which affects the dielectric constant of the water, and (iii) change in the rate of TiO2-promoted cytochrome c reduction when water was replaced with D2O. The TiO2-promoted photo-reduction of cytochrome c was reverted by peroxides. Cytochrome c incorporated in the titanate nanotubes was also reversibly reduced under irradiation, as confirmed by EPR and UV-visible spectroscopy.

ChemInform Abstract: Ru(bpy)3-Based Artificial Receptors Toward a Protein Surface: Selective Binding and Efficient Photoreduction of Cytochrome c.

The binding properties of a series of Ru(bpy)3 complexes to cytochrome c are described; these compounds act as selective cytochrome c receptors and one derivatie is shown to be a photodriven modulator of the cytochrome c redox state.

Transient and Stationary Spectroscopy of Cytochrome c: Ultrafast Internal Conversion Controls Photoreduction

Photoreduction of cytochrome c (Cyt c) has been reinvestigated using femtosecond-to-nanosecond transient absorption and stationary spectroscopy. Femtosecond spectra of oxidized Cyt c, recorded in the probe range 270-1000 nm, demonstrate similar evolution upon 266 or 403 nm excitation: an ultrafast 0.3 ps internal conversion followed by a 4 ps vibrational cooling. Late transient spectra after 20 ps, from the cold ground-state chromophore, reveal a small but measurable signal from reduced Cyt c. The yield phi for Fe3+-->Fe2+ photoreduction is measured to be phi(403) = 0.016 and phi(266) = 0.08 for 403 and 266 nm excitation. These yields lead to a guess of the barrier E(f)(A) = 55 kJ mol(-1) for thermal ground-state electron transfer (ET). Nanosecond spectra initially show the typical absorption from reduced Cyt c and then exhibit temperature-dependent sub-microsecond decays (0.5 micros at 297 K), corresponding to a barrier E(A)(b) = 33 kJ mol(-1) for the back ET reaction and a reaction energy DeltaE = 22 kJ mol(-1). The nanosecond transients do not decay to zero on a second time scale, demonstrating the stability of some of the reduced Cyt c. The yields calculated from this stable reduced form agree with quasistationary reduction yields. Modest heating of Cyt c leads to its efficient thermal reduction as demonstrated by differential stationary absorption spectroscopy. In summary, our results point to ultrafast internal conversion of oxidized Cyt c upon UV or visible excitation, followed by Fe-porphyrin reduction due to thermal ground-state ET as the prevailing mechanism.

3P113 チトクロム酸化酵素のX線による還元効果の構造解析(ヘム蛋白質))

3P113 チトクロム酸化酵素のX線による還元効果の構造解析(ヘム蛋白質))

Isolation and biophysical studies of natural eumelanins: applications of imaging technologies and ultrafast spectroscopy.

The major pigments found in the skin, hair, and eyes of humans and other animals are melanins. Despite significant research efforts, the current understanding of the molecular structure of melanins, the assembly of the pigment within its organelle, and the structural consequences of the association of melanins with protein and metal cations is limited. Likewise, a detailed understanding of the photochemical and photophysical properties of melanins has remained elusive. Many types of melanins have been studied to date, including natural and synthetic model pigments. Such studies are often contradictory and to some extent the diversity of systems studied may have detracted from the development of a basic understanding of the structure and function of the natural pigment. Advances in the understanding of the structure and function of melanins require careful characterization of the pigments examined so as to assure the data obtained may be relevant to the properties of the pigment in vivo. To address this issue, herein the influence of isolation procedures on the resulting structure of the pigment is examined. Sections describing the applications of new technologies to the study of melanins follow this. Advanced imaging technologies such as scanning probe microscopies are providing new insights into the morphology of the pigment assembly. Recent photochemical studies on photoreduction of cytochrome c by different mass fraction of sonicated natural melanins reveal that the photogeneration of reactive oxygen species (ROS) depends upon aggregation of melanin. Specifically, aggregation mitigates ROS photoproduction by UV-excitation, suggesting the integrity of melanosomes in tissue may play an important role in the balance between the photoprotective and photodamaging behaviors attributed to melanins. Ultrafast laser spectroscopy studies of melanins are providing insights into the time scales and mechanisms by which melanin dissipates absorbed light energy.

A new catalytic site in the cytochrome b6f complex of Chlamydomonas reinhardtii

The Q-cycle model for the operation of the cytochrome b6 f complex is based on the oxidant-induced-reduction phenomenon and proposes that the obligatory sequence of events that, after a flash, leads to the reduction of cytochrome b is: 1) oxidation of cytochrome f, 2) oxidation of the FeS center by cytochrome f (i.e. re-reduction of cytochrome f), 3) oxidation of PQH2 to semiquinone by the FeS center, and 4) reduction of the cytochrome b chain by semiquinone. Therefore, cytochrome b (s) should be reduced no faster than cytochrome f. C. reinhardtii mutants of cytochrome f show inhibited rates of cytochrome f re-reduction (a 100 fold range; t1/2 for wt is ~ 3 ms). Instead, the photoreduction of cytochrome b in those mutants is slightly or not inhibited (t1/2 for wt is ~ 3 ms). Thus, the reduction of cytochrome b can precede the re-reduction of cytochrome f, violating the Q-cycle tenets. Besides, the re-reductions of P700 and of plastocyanin that are sensitive to Qo-site inhibitors are not correspondingly inhibited in the mutants and are much faster than the re-reduction of cytochrome f (t1/2= 700 m s for P700); this is also true in the wt. Therefore, although cytochrome f shows redox activity, it is not an obligatory pathway for electrons flowing through the cytochrome b6 f complex from PQH2 to Photosystem I. Steady-state photosynthetic electron transfer activities support this conclusion. We propose that the oxidizing equivalents from Photosystem I can be delivered to the high potential chain of the cytochrome b6 f complex both at the level of cytochrome f and, independently, at another site that is connected to the Qo site, possibly the FeS center. (1) J.Biol.Chem. (2001), accepted.

Photoinduced intracomplex electron transfer between cytochrome c oxidase and TUPS-modified cytochrome c.

A novel method for initiating intramolecular electron transfer in cytochrome c oxidase is reported. The method is based upon photoreduction of cytochrome c labeled with thiouredopyrene-3,6, 8-trisulfonate in complex with cytochrome oxidase. The thiouredopyrene-3,6,8-trisulfonate-labeled cytochrome c was prepared by incubating the thiol reactive form of the dye with yeast iso-1-cytochrome c, containing a single cysteine residue. Laser pulse excitation of a stoichiometrical complex between thiouredopyrene-3,6,8-trisulfonate-cytochrome c and bovine heart cytochrome oxidase at low ionic strength resulted in the reduction of cytochrome c by the excited form of thiouredopyrene-3,6, 8-trisulfonate and subsequent intramolecular electron transfer from the reduced cytochrome c to cytochrome oxidase. The maximum efficiency by a single laser pulse resulted in the reduction of approximately 17% of cytochrome a, and was achieved only at a 1 : 1 ratio of cytochrome c to cytochrome oxidase. At higher cytochrome c to cytochrome oxidase ratios the heme a reduction was strongly suppressed.

Light induced redox reactions involving mammalian ferritin as photocatalyst

Under excitation by visible light the iron storage protein ferritin catalyses the reduction of cytochrome c and viologens as well as the oxidation of carboxylic acids, thiol compounds, and sulfite. The photochemically active element of ferritin is its mineral ferrihydrite semiconductor core. Band-gap excitation of these microcrystals leads to generation of electron-hole pairs that are sufficiently long-lived and reactive to engage in redox reactions with components of the medium. Photoreduction of cytochrome c and viologens occurs due to electron transfer from the conduction band of the iron oxide cluster through the protein shell surrounding the ferritin core. Laser photolysis coupled with time-resolved kinetics spectroscopy showed the electron transfer to propylviologen sulfonate to proceed in the microsecond time range. In the absence of electron acceptor at pH < 7, light excitation results in photodissolution of the iron oxide cluster with concomitant formation of Fe(II). These novel findings concerning the photocatalytic activity of ferritin with its inherent biological implications are discussed.

Photoreduction of cytochrome c with zinc protoporphyrin reconstituted myoglobin (ZnPPMb)

Photoreduction of cytchrome c (cyt. c) proceeded by irradiation of the visible light in the reaction system consisting of EDTA, ZnPPMb and cyt. c. About 90% of cyt. c was reduced in 20 min of irradiation of the light. Photoinduced electron transfer occurs between ZnPPMb and cyt. c in the above system. Laser flash photolysis showed that the oxidative quenching of 3ZnPPMb∗ by the oxidized cyt. c took place and the quenching rate constant was 8.5 × 10 5 M −1s −1.

Photoreduction of cytochrome c using a RU(phen)2(CN)- label at His33: New methods for studying intra and intermolecular electron transfer in metalloproteins.

Photoreduction of cytochrome c using a RU(phen)2(CN)- label at His33: New methods for studying intra and intermolecular electron transfer in metalloproteins.

Resonance Raman study on photoreduction of cytochrome c oxidase: distinction of cytochromes a and a3 in the intermediate oxidation states.

Occurrence of photoreduction of bovine cytochrome c oxidase was confirmed with the difference absorption spectra and oxygen consumption measurements for the enzyme irradiated with laser light at 406.7, 441.6, and 590 nm. The resonance Raman spectra were obtained under the same experimental conditions as those adopted for the measurements of oxygen consumption and difference absorption spectra. The photoreduction was more effective upon irradiation at shorter wavelengths and was irreversible under anaerobic conditions. However, upon aeration into the cell, the original oxidized form was restored. It was found that aerobic laser irradiation produces a photo steady state of the catalytic dioxygen reduction and that the Raman scattering from this photo steady state probes cytochrome a2+ and cytochrome a3(3)+ separately upon excitations at 441.6 and 406.7 nm, respectively. The enzyme was apparently protected from the photoreduction in the spinning cell with the spinning speed between 1 and 1500 rpm. These results were explained satisfactorily with the reported rate constant for the electron transfer from cytochrome a to cytochrome a3 (0.58 s-1) and a comparable photoreduction rate of cytochrome a. The anaerobic photoreduction did give Raman lines at 1666 and 214 cm-1, which are characteristic of the ferrous high-spin cytochrome a3(2)+, but they were absent under aerobic photoreduction. The formyl CH = O stretching mode of the a3 heme was observed at 1671 cm-1 for a2+a3(2)+CO but at 1664 cm-1 for a2+a3(2)+CN-, indicating that the CH = O stretching frequency reflects the pi back-donation to the axial ligand similar to the oxidation state marker line (v4).

A redox study of the electron transport pathway responsible for generation of the slow electrochromic phase in chloroplasts

The amplitude of the slow phase of the electrochromic bandshift and the dark redox state of cytochrome b 6, as well as its flash-induced turnover, have been measured as a function of ambient redox potential between +200 and −200 mV. Formation of a quinol-like donor with an E m,7 = +100 ± 10 mV is required for generation of the slow phase. 80–100% of the amplitude of this signal with a t 1 2 = 3−4 ms is observed at −200 mV where cytochrome b 6 was almost fully reduced ( E m,7 of dark and flash-induced photoreduction was −30 mV and −75 mV, respectively). The change in the photoreduction of cytochrome b 6 above 0 mV had an E m,7 of +50 mV, about 50 mV more negative than the midpoint at this pH for the onset of the slow electrochromic change. At potentials below −140mV the amplitude of b 6 photoreduction becomes small or negligible. The nature of the cytochrome b 6 photoresponse is changed at potentials below −140 mV from a net photoreduction with a t 1 2 = ≲ 1 ms to a photooxidation with a t 1 2 = 15−20 ms that is substantially slower than the electrochromic band-shift with a t 1 2 = 3−4 ms . It is concluded that the slow electrochromic phase probably does not arise from a mechanism involving a turnover of cytochrome b 6. From consideration of the possible flash-induced electron-transfer steps and alternative mechanisms for generation of the slow phase, it is suggested that it may arise from a redox-linked H + pump involving the high potential iron-sulfur protein.

Photoreactions of cytochrome b6 in systems using resolved chloroplast electron-transfer complexes

Photoreactions of cytochrome b 6 have been studied using resolved chloroplast electron-transfer complexes. In the presence of Photosystem (PS) II and the cytochrome b 6- f complex, photoreduction of the cytochrome can be observed. No soluble components are required for this reaction. Cytochrome b 6 photoreduction was found to be inhibited by quinone analogs, which inhibit at the Rieske iron-sulfur center of the cytochrome complex, by the addition of ascorbate and by depletion of the Rieske center and bound plastoquinone from the cytochrome complex. Photoreduction of cytochrome b 6 can also be demonstrated in the presence of the cytochrome complex and PS I. This photoreduction requires plastocyanin and a low-potential electron donor, such as durohydroquinone. Cytochrome b 6 photoreduction in the presence of PS I is inhibited by quinone analogs which interact with the Rieske iron-sulfur center. These results are discussed in terms of a Q-cycle mechanism in which plastosemiquinone serves as the reductant for cytochrome b 6 via an oxidant-induced reductive pathway.

Chemical modification of spinach ferredoxin: Evidence for the involvement of a complex between ferredoxin and ferredoxin:NADP oxidoreductase in NADP photoreduction

Spinach ferredoxin was modified chemically with trinitrobenzene sulfonic acid (TNBS), a reagent which reacts specifically with amino groups. The trinitrophenylated ferredoxin (TNP-Fd) can accept electrons from Photosystem I as indicated by its full activity in the photoreduction of cytochrome c . The modified protein is inactive, however, in the photoreduction of NADP and cannot form a complex with the flavoprotein, ferredoxin: NADP oxidoreductase. The data presented indicate that the inactivity of the modified protein is the result of modification of a single amino group.

FLAVIN‐MEDIATED PHOTOREACTIONS IN ARTIFICIAL SYSTEMS: A POSSIBLE MODEL FOR THE BLUE‐LIGHT PHOTORECEPTOR PIGMENT IN LIVING SYSTEMS*

Abstract— The photoreduction of cytochrome c and the photostimulation of oxygen uptake were studied in solutions of flavin and cytochrome as a possible model system for similar photoreactions which have been observed in vivo. Light causes the photoreduction of the flavin. Under aerobic conditions the photoreduced flavin reacts with oxygen to form the superoxide anion which in turn can reduce cytochrome c. Dismutation of the superoxide anions forms hydrogen peroxide which mediates the dark oxidation of the photoreduced cytochrome. Superoxide formation and dismutation also account for the light‐induced oxygen uptake. Action spectra confirm the role of flavin in the photoreduction of cytochrome c and the photostimulation of oxygen uptake. Under anaerobic conditions the photoreduced flavin reduces cytochrome c directly. In the presence of an electron donor only catalytic amounts of flavin are required. In the absence of an added electron donor flavin itself can act as the electron donor if substrate amounts are present. Azide inhibits all of these flavin‐mediated photoresponses. Azide also inhibits the photoreduction of cytochrome b which occurs in the mycelium of Newospora.

Studies on electron transport associated with Photosystem I. I. Functional site of plastocyanin: Inhibitory effects of HgCl 2 on electron transport and plastocyanin in chloroplasts

1. Incubation of chloroplasts with HgCl 2 at a molar ratio of HgCl 2 to chlorophyll of about unity, induced a complete inhibition of the methyl viologen Hill reaction, as well as methyl viologen photoreduction with reduced 2,6-dichlorophenolindophenol (DCIP) as electron donor. Photooxidation of cytochrome ƒ was similarly sensitive towards HgCl 2, whereas photooxidation of P700 was resistant to the poison. Photoreduction of cytochrome ƒ and light-induced increase in fluorescence yield were enhanced by the HgCl 2 treatment of chloroplasts. 2. Photooxidation of reduced yeast cytochrome c catalyzed by Photosystem I particles in the presence of plastocyanin was also suppressed by preincubation with HgCl 2. The inhibition was recovered by adding more plastocyanin, but not by addition of the particles, to the reaction mixture. 3. Incubation of reduced plastocyanin with HgCl 2 caused a bleaching of the blue color of the protein, whereas the oxidized protein was resistant to HgCl 2. A similar contrasting difference in sensitivity towards HgCl 2 was found in the Hill reaction in the absence and presence of ferricyanide. 4. Sonic treatment released plastocyanin from the untreated chloroplasts, whereas no plastocyanin was detected in the extract of the HgCl 2-treated chloroplasts. However, plastocyanin could be reconstructed by dialyzing the extract of the HgCl 2-treated chloroplasts against cysteine solution and then CuSO 4 solution. 5. The role of plastocyanin in the photosynthetic electron transport is discussed.

Photoreduction of cytochrome b559 in a Photosystem-II subchloroplast particle

A Photosystem-II reaction-center particle derived from spinach chloroplasts by Triton treatment contains only one kind of cytochrome, namely, cytochrome b 559, in the amount of slightly more than 2 per 100 total chlorophyll molecules. Cytochrome b 559 is present in the oxidized form, has a standard redox potential of 58 mV, and undergoes photoreduction. Photoreduction of cytochrome b 559 and its re-oxidation in the dark can take place in the Photosystem-II particle without the addition of any exogenous electron donors such as diphenylcarbazide. The same reaction can even be better observed under anaerobic condition when oxidative photobleaching of bulk chlorophyll is prevented. Whereas the photoreduction of 2,6-dichlorophenolindophenol catalyzed by the Photosystem-II particle and supported by diphenylcarbazide is completely inhibited by 2 · 10 −5 M 3-(3,4-dichlorophenyl)-1,1-dimethylura (DCMU) or carbonylcyanide- m-chlorophenylhydrazone, the photoreduction of cytochrome b 559 is inhibited only 40 and 20%, respectively. The quantum efficiency of cytochrome b 559 photoreducation is near unity. An Arrhenius plot yields an activation energy of 7.5 kcal/mole. The cytochrome reaction ceases when the reaction mixture becomes frozen. No photooxidation of cytochrome b 559 at 77°K has been observed even when it was pre-reduced chemically. Observations made thus far support the notion that cytochrome b 559 is reduced by a reducing component formed in the electron-transport chain and re-oxidized by an oxidizing component formed on the oxidizing side of Photosystem-II. Thus, the light-induced redox reactions of cytochrome b 559 form a small cycle around Photosystem-II. The nature for the lack of inhibition of cytochrome b 559 photoreduction by DCMU is not yet clear.

PHOTOREACTIONS OF CHLOROPLASTS AND CHLOROPHYLL A WITH HYDROQUINONES AND QUINONES: COUPLED PHOTOREDUCTION OF CYTOCHROME C.

PHOTOREACTIONS OF CHLOROPLASTS AND CHLOROPHYLL A WITH HYDROQUINONES AND QUINONES: COUPLED PHOTOREDUCTION OF CYTOCHROME C.