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At least 883 records · Page 49Linked to original sources
PubMed1985

Microsomal cytochrome P-450 "handprints": five fractions from anion-exchange high-pressure liquid chromatography provide a rapid preliminary screen for selectivity in the induction and destruction of rat hepatic cytochrome P-450 subpopulations.

Anion-exchange HPLC was used to monitor changes in rat hepatic cytochrome P-450 fractions resulting from animal exposure to several inducing and hepatotoxic agents. The column eluant was divided into five fractions and the cytochrome P-450 content in each was determined. Induction of hepatic cytochrome P-450 following 2,5-hexanedione and SKF 525-A pretreatment of animals showed an induction pattern in the five-fraction "handprint" similar to that produced by phenobarbital. The patterns of induction following beta-naphthoflavone, clofibrate, and isosafrole pretreatments were unique in comparison to each other and also compared to that of phenobarbital. The pattern of cytochrome P-450 loss after allyl alcohol was similar to that seen after cobalt chloride treatment, but differed from that seen following acetaminophen administration. Five-fraction quantitation of the eluant from anion-exchange HPLC of microsomes appears to be a useful technique to screen for and convey the effects of previously uncharacterized inducers and the effects of hepatotoxic agents or heme synthesis inhibitors on rat hepatic cytochrome P-450 subpopulations.

Animals↗
PubMed1986

Localization of cytochrome C oxidase and cytochrome C peroxidase in mitochondria of Hymenolepis diminuta (Cestoda).

The intramitochondrial localization of cytochrome c oxidase and cytochrome c peroxidase in adult Hymenolepis diminuta was investigated. Mitochondria were fractionated into inner membrane, outer membrane, intermembrane space and matrix and the efficacy of fractionation was monitored employing marker enzymes. Cytochrome c oxidase was associated with the mitochondrial inner membrane. Whereas 55% of the cytochrome c peroxidase activity was in the matrix, 32% of the activity was in the intermembrane space fraction. Based upon the distribution of marker enzymes, a dual compartmentalization of cytochrome c peroxidase is apparent in H. diminuta mitochondria.

Animals↗
PubMed2004

Novel cyanide inhibition at cytochrome c1 of Rhodobacter capsulatus cytochrome bc1.

Oxidized cytochrome c(1) in photosynthetic bacterium Rhodobacter capsulatus cytochrome bc(1) reversibly binds cyanide with surprisingly high, micromolar affinity. The binding dramatically lowers the redox midpoint potential of heme c(1) and inhibits steady-state turnover activity of the enzyme. As cytochrome c(1), an auxiliary redox center of the high-potential chain of cytochrome bc(1), does not interact directly with the catalytic quinone/quinol binding sites Q(o) and Q(i), cyanide introduces a novel, Q-site independent locus of inhibition. This is the first report of a reversible inhibitor that manipulates the energetics and electron transfers of the high-potential redox chain of cytochrome bc(1), while maintaining quinone substrate catalytic sites in an intact form.

Binding Sites↗
PubMed2006

A comparative kinetic analysis of the reactivity of plant, horse, and human respiratory cytochrome c towards cytochrome c oxidase.

Two synthetic genes coding for human and Arabidopsis cytochrome c, respectively, have been designed and constructed, and the recombinant proteins have been over-expressed in Escherichia coli cells. Thus a comparative analysis of the two heme proteins, including horse cytochrome c as a reference, has been performed. In addition to their physico-chemical properties, the redox behavior of the three proteins has been analyzed by following the kinetics of both their reduction by flavin semiquinones (lumiflavin, riboflavin, and FMN) and oxidation by cytochrome c oxidase. The resulting data indicate that the accessibility and electrostatic charge of the active site do not differ in a significant way among the three proteins, but human cytochrome c exhibits some intriguing differences when interacting with cytochrome c oxidase that could be related to the amino acid changes underwent by the latter along evolution.

Animals↗
PubMed2006

Desulfovibrio desulfuricans G20 tetraheme cytochrome structure at 1.5 Angstrom and cytochrome interaction with metal complexes.

The structure of the type I tetraheme cytochrome c(3) from Desulfovibrio desulfuricans G20 was determined to 1.5 Angstrom by X-ray crystallography. In addition to the oxidized form, the structure of the molybdate-bound form of the protein was determined from oxidized crystals soaked in sodium molybdate. Only small structural shifts were obtained with metal binding, consistent with the remarkable structural stability of this protein. In vitro experiments with pure cytochrome showed that molybdate could oxidize the reduced cytochrome, although not as rapidly as U(VI) present as uranyl acetate. Alterations in the overall conformation and thermostability of the metal-oxidized protein were investigated by circular dichroism studies. Again, only small changes in protein structure were documented. The location of the molybdate ion near heme IV in the crystal structure suggested heme IV as the site of electron exit from the reduced cytochrome and implicated Lys14 and Lys56 in binding. Analysis of structurally conserved water molecules in type I cytochrome c(3) crystal structures identified interactions predicted to be important for protein stability and possibly for intramolecular electron transfer among heme molecules.

Binding Sites↗
PubMed2002

Electron transfer between yeast cytochrome bc(1) complex and cytochrome c: a structural analysis.

The structure of the complex between cytochrome c (CYC) and the cytochrome bc(1) complex (QCR) from yeast crystallized with an antibody fragment has been recently determined at 2.97 A resolution [Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 2800]. CYC binds to subunit cytochrome c(1) of the enzyme stabilized by hydrophobic interactions surrounding the heme crevices creating a small, compact contact site. A central cation-pi interaction is an important and conserved feature of CYC binding. Peripheral patches with highly conserved complementary charges further stabilize the enzyme-substrate complex by long-range electrostatic forces and may affect the orientation of the substrate. Size and characteristics of the contact site are optimal for a transient electron transfer complex. Kinetic data show a bell-shaped ionic strength dependence of the cytochrome c reduction with a maximum activity near physiological ionic strength. The dependence is less pronounced in yeast compared to horse heart CYC indicating less impact of electrostatic interactions in the yeast system. Interestingly, a local QCR activity minimum is found for both substrates at 120-140 mM ionic strength. The architecture of the complex results in close distance of both c-type heme groups allowing the rapid reduction of cytochrome c by QCR via direct heme-to-heme electron transfer. Remarkably, CYC binds only to one of the two possible binding sites of the homodimeric complex and binding appears to be coordinated with the presence of ubiquinone at the Q(i) site. Regulatory aspects of CYC reduction are discussed.

Crystallization↗
PubMed2002

Structural changes are induced in human neutrophil cytochrome b by NADPH oxidase activators, LDS, SDS, and arachidonate: intermolecular resonance energy transfer between trisulfopyrenyl-wheat germ agglutinin and cytochrome b(558).

Anionic amphiphiles such as sodium- and lithium dodecyl sulfate (SDS, LDS), or arachidonate (AA) initiate NADPH oxidase and proton channel activation in cell-free systems and intact neutrophils. To investigate whether these amphiphiles exert allosteric effects on cytochrome b, trisulfopyrenyl-labeled wheat germ agglutinin (Cascade Blue-wheat germ agglutinin, CCB-WGA) was used as an extrinsic fluorescence donor for resonance energy transfer (RET) to the intrinsic heme acceptors of detergent-solubilized cytochrome b. In solution, cytochrome b complexed with the CCB-WGA causing a rapid, saturable, carbohydrate-dependent quenching of up to approximately 55% of the steady-state fluorescence. Subsequent additions of SDS, LDS, or AA to typical cell-free oxidase assay concentrations completely relaxed the fluorescence quenching. The relaxation effects were specific, and not caused by dissociation of the CCB-WGA-cytochrome b complex or alterations in the spectral properties of the chromophores. In contrast, addition of the oxidase antagonist, arachidonate methyl ester, caused an opposite effect and was able to partially reverse the activator-induced relaxation. We conclude that the activators induce a cytochrome b conformation wherein the proximity or orientation between the hemes and the extrinsic CCB fluorescence donors has undergone a significant change. These events may be linked to NADPH oxidase assembly and activation or proton channel induction.

Arachidonic Acid↗
PubMed1987

ADP increases the affinity for cytochrome c by interaction with the matrix side of bovine heart cytochrome c oxidase.

The effect of intraliposomal ADP and ATP on the kinetics of cytochrome c oxidation in reconstituted bovine heart cytochrome c oxidase was measured by the photometric and polarographic method: 1. Intraliposomal ADP decreases and intraliposomal ATP increases the Km for cytochrome c when measured by the photometric assay under uncoupled conditions. 2. The above described effects are not obtained when the kinetics are measured with the polarographic assay. 3. Extraliposomal ATP increases the Km for cytochrome c similar to intraliposomal ATP, but this effect is measured with both methods of assay. 4. Under coupled conditions only a small decrease of the Km for cytochrome c by intraliposomal ADP is found.

Adenosine Diphosphate↗
PubMed1995

Effect of ionic strength on the kinetic mechanism and relative rate limitation of steps in the model NADPH-cytochrome P450 oxidoreductase reaction with cytochrome c.

Although the kinetic mechanism of the NADPH-cytochrome P450 oxidoreductase (P450R) reaction with cytochrome c3+ has been determined at 850 mM ionic strength [Sem, D.S., & Kasper, C. B. (1994) Biochemistry 33, 12012-12021], this mechanism is no longer valid at lower ionic strength. At 850 mM ionic strength, the mechanism is two-site ping-pong, and reaction at the electron acceptor site is itself ping-pong. As the ionic strength is decreased below 850 mM, the initial velocity profiles begin to show curvature when cytochrome c3+ is the varied substrate. These data are consistent with a mechanism that is still two-site ping-pong, but now with random sequential binding of two molecules of cytochrome c3+ at the electron acceptor site. Decreasing ionic strength also causes a change in rate-limiting steps, with (V/K)cytc and (V/K)NADPH increasing while Vmax decreases (below 500 mM ionic strength). These results are consistent with favorable ionic interactions being important for binding NADPH and cytochrome c3+ and with product (NADP+) release becoming the rate-limiting step in Vmax at low ionic strength. Vmax decreases significantly at higher ionic strength (> 500 mM), while (V/K)NADPH decreases only slightly. The DV isotope effect is largest (2.4) at 500 mM ionic strength but decreases at both low and high ionic strength as steps other than hydride transfer become more rate-limiting. D(V/K)NADPH also decreases at both low and high ionic strength, but to a lesser extent than DV.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

Interaction of cytochrome c with cytochrome c oxidase: an NMR study on two soluble fragments derived from Paracoccus denitrificans.

The functional interactions between the various components of the respiratory chain are relatively short-lived, thus allowing high turnover numbers but at the same time complicating the structural analysis of the complexes. Chemical shift mapping by NMR spectroscopy is a useful tool to investigate such transient contacts, since it can monitor changes in the electron-shielding properties of a protein as the result of temporary contacts with a reaction partner. In this study, we investigated the molecular interaction between two components of the electron-transfer chain from Paracoccus denitrificans: the engineered, water-soluble fragment of cytochrome c(552) and the Cu(A) domain from the cytochrome c oxidase. Comparison of [(15)N,(1)H]-TROSY spectra of the [(15)N]-labeled cytochrome c(552) fragment in the absence and in the presence of the Cu(A) fragment showed chemical shift changes for the backbone amide groups of several, mostly uncharged residues located around the exposed heme edge in cytochrome c(552). The detected contact areas on the cytochrome c(552) surface were comparable under both fully reduced and fully oxidized conditions, suggesting that the respective chemical shift changes represent biologically relevant protein-protein interactions.

Bacterial Proteins↗
PubMed2006

The role of an extra fragment of cytochrome b (residues 309-326) in the cytochrome bc1 complex from Rhodobacter sphaeroides.

In bacterial cytochrome b of the cytochrome bc(1) complex, there is an extra fragment located between the amphipathic helix ef and the transmembrane helix F compared to the mitochondrial counterparts. In this work, mutants at various positions of this extra fragment were generated in Rhodobacter sphaeroides in an effort to investigate its specific role in the bacterial bc(1) complex. The total deletion [cytb-Delta(309-326)] and alanine substitution [cytb-(309-326)A] mutant complexes have about 20% of the bc(1) activity found in the wild-type complex. Mutant complexes of cytb-(309-311)A, cytb-(312-314)A, cytb-(315-317)A, cytb-(318-321)A, cytb-(322-323)A, cytb-(324-326)A, cytb-(F323A), and cytb-(S322A) have respectively 87%, 85%, 89%, 100%, 32%, 90%, 100%, and 32% of the bc(1) activity, indicating that the S322 of cytochrome b is important. EPR spectral analysis reveals that the [2Fe-2S] cluster in the cytb-(S322A) mutant complex has a broadened and shifted g(x)() signal (g = 1.76). The rate of superoxide anion (O(2)(*)(-)) generation is 4 times higher in the cytb-(S322A) mutant complex than in the wild-type or mutant complexes of S322T, S322Y, or S322C. These results support the idea that alanine substitution at S322 of cytochrome b causes conformational changes at the Q(o) site by weakening the binding between cytochrome b and ISP through hydrogen bonding provided by the hydroxyl group of this residue. This change facilitates electron leakage from the Q(o) site for reaction with molecular oxygen to form superoxide anion, thus decreasing bc(1) activity.

Amino Acid Sequence↗
PubMed2000

Supramolecular complex of cytochrome c with lariat ether: solubilization, redox behavior and catalytic activity of cytochrome c in methanol.

A variety of lariat ethers were employed to solubilize water-soluble cytochrome c in methanol, in which alcohol, ether, ester, amine, and amide functionalities were attached as cation-ligating side arms to 18-crown-6, 15-crown-5, and 12-crown-4 rings. Among these lariat ethers, the alcohol-armed 18-crown-6 derivative offered the highest solubilization efficiency for cytochrome c via supramolecular complexation. The resulting cytochrome c-lariat ether complexes were electrochemically and spectroscopically characterized and confirmed to have redox-active heme structures of 6-coordinate low-spin population in methanol. Some of them catalyzed the oxidation of pinacyanol chloride with hydrogen peroxide in methanol and exhibited higher activities than unmodified cytochrome c and its poly(ethylene glycolated) derivative. Since the supramolecular complexation between lariat ether and cytochrome c includes extremely simple procedures, it provides a facile preparation method of effective biocatalysts working in organic solvents from metalloproteins.

Carbocyanines↗
PubMed2001

Pinocytic loading of cytochrome c into intact cells specifically induces caspase-dependent permeabilization of mitochondria: evidence for a cytochrome c feedback loop.

Previous studies introduced cytochrome c into intact cells via the disruptive techniques of microinjection or electroporation to provide support for the hypothesis that, in whole cells, cytochrome c release from mitochondria triggers caspase activation and other degradative changes. However, the types of measurements that could be undertaken with these techniques was limited. We used the simple and relatively gentle technique of pinocytic loading to demonstrate that, in intact cells, cytosolic cytochrome c specifically induced activation of caspase-3- and -9-like enzymes, and a loss of mitochondrial polarization coincident with an increase in mitochondrial permeability. Our results support the prediction from in vitro studies that activation of caspases-3 and -9 is downstream of cytochrome c release and provide the first direct evidence that, in whole cells, cytochrome c-dependent caspase-activation can exert a feedback effect to elicit mitochondrial permeabilization and collapse of the mitochondrial trans-membrane potential.

Apoptosis↗
PubMed1979

The oxidation-reduction potentials of cytochrome o + c4 and cytochrome o purified from Azotobacter vinelandii.

Oxidation-reduction titrations of Azotobacter vinelandii cytochrome o + c4 and cytochrome o were performed with simultaneous potential and absorbance measurements under anaerobic conditions. Cytochrome c4 has a midpoint potential (Em, 7.4) of 260mV and purified cytochrome o has an Em, 7.4 of -18mV. Little change in the midpoint potential of cytochrome o was observed when titrated in the pH range 6.2--9.8.

Azotobacter↗
PubMed1983

CO-binding c-type cytochromes and a high-potential cytochrome c in Nitrosomonas europaea.

The purification of two soluble CO-binding cytochromes c from Nitrosomonas europaea is described. Cytochrome cCO-550 ran on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an apparent Mr of 32 000, whereas for cytochrome cCO-552 the apparent Mr was 16 000. Redox potentials (Em, 7) were determined as +140 and -50mV respectively. Cytochrome cCO-550 was co-purified with a cytochrome c-553, for which an unusually high redox potential of +450mV was measured. These latter components were not resolved by gel-filtration chromatography or electrophoresis under denaturing conditions.

Carbon Monoxide↗
PubMed1985

Effects of detergents and cytochrome c binding on scalar and vectorial proton ejection by proteoliposomes containing cytochrome oxidase.

The detergent lauryl maltoside abolishes respiratory control and proton ejection by cytochrome c oxidase-containing proteoliposomes over a narrow concentration range. Expression of cryptic activity (inward-facing oxidase) is released over the same concentration range. Catalytic functions (Vmax. and Km) of the enzyme are not changed by the detergent. Lipid micelles containing detergent bind approximately the same amount of cytochrome c as do vesicles containing an equivalent amount of lipid. Uncoupler-insensitive proton release is seen when proteoliposomes are pulsed with ferrocytochrome c at low ionic strength. Such uncoupler-insensitive acidification is not seen at higher ionic strength, nor with oxygen pulses of anaerobic solutions previously incubated with cytochrome c. Vesicles at low ionic strength catalyse cytochrome c autoxidation; this process can mimic proton re-equilibration in systems that have pumped protons from inside to the bulk phase. Proton re-equilibration following a pulse of cytochrome c or oxygen is multiphasic. The slowest phases are attributed to vesicle heterogeneity, some internal alkali being retained within vesicles of low intrinsic proton permeability. This can be overcome by the addition of either very low levels of carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone or high levels of valinomycin.

Cytochrome c Group↗
PubMed2001

Assembly of cytochrome c oxidase: what can we learn from patients with cytochrome c oxidase deficiency?

Cytochrome c oxidase is an intricate metalloprotein that transfers electrons from cytochrome c to oxygen in the last step of the mitochondrial respiratory chain. It uses the free energy of this reaction to sustain a transmembrane electrochemical gradient of protons. Site-directed mutagenesis studies of bacterial terminal oxidases and the recent availability of refined crystal structures of the enzyme are rapidly expanding the understanding of the coupling mechanism between electron transfer and proton translocation. In contrast, relatively little is known about the assembly pathway of cytochrome c oxidase. Studies in yeast have indicated that assembly is dependent on numerous proteins in addition to the structural subunits and prosthetic groups. Human homologues of a number of these assembly factors have been identified and some are now known to be involved in disease. To dissect the assembly pathway of cytochrome c oxidase, we are characterizing tissues and cell cultures derived from patients with genetically defined cytochrome c oxidase deficiency, using biochemical, biophysical and immunological techniques. These studies have allowed us to identify some of the steps of the assembly process.

Animals↗
PubMed1998

Cytochrome-c-binding site on cytochrome oxidase in Paracoccus denitrificans.

To monitor the docking site for cytochrome c on cytochrome oxidase from Paracoccus denitrificans, a series of site-directed mutants in acidic residues exposed on the three largest subunits was constructed, and the purified enzymes were assayed for their steady-state kinetic parameters, their ionic strength dependence, and their fast electron entry kinetics by stopped-flow measurements. Increasing the ionic strength, the maximum of the bell-shaped dependence of the steady-state rate observed for wild type shifts the maximum to lower ionic strength in most of the mutants. The Km determined in steady-state experiments under different conditions is largely increased for most of the subunit II and one of the subunit I mutants, giving evidence that binding is impaired, whereas subunit III residues do not seem to contribute significantly. In addition, the bimolecular rate constant for cytochrome c oxidation under pre-steady state conditions was measured using stopped flow spectroscopy. Taken together, the results demonstrate that the initial interaction of cytochrome c and oxidase is mediated through glutamates and aspartates mainly located in subunit II. The crystal structure of oxidase reveals that the participating residues are clustered, creating an extended, negatively charged patch. We propose this clustering to be a decisive factor in the recognition of positively charged patches on the surface of cytochrome c.

Binding Sites↗