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1.
In order to distinguish between the regulatory effects of oxygen tension and light intensity on cytochrome c oxidase protein and enzymatic activity cells of Rhodobacter capsulatus were shifted from phototrophic (anaerobic, light) growth to aerobic-light, aerobic-dark and to anaerobic-dark conditions, respectively. During shift-experiments the formation of oxidase protein and regulation of oxidase activity was followed by immunological and enzymatic means. The results support the idea, that the formation of oxidase protein is regulated by oxygen tension and light intensity changes, whereas the regulation of oxidase activity seems only to be correlated to the oxygen tension. A DNA sequence involved in the oxygen-dependent regulation of cytochrome oxidase could be identified in the regulation-deficient oxidase mutant H41 of R. capsulatus. Immunological investigations of cytochrome c
2 from mutant H41 demonstrated at the same time the participation of the c
2-polypeptide in the regulation of cytochrome c oxidase.Abbreviations Bchl
bacteriochlorophyll
- CIE
crossed immuno-electrophoresis
- DMSO
dimethyl sulfoxide 相似文献
2.
Both the soluble cytochrome c2 and the membrane-bound cytochrome cy act as secondary electron carriers in photoinduced cyclic electron transfer chain of Rhodobacter capsulatus [Jenney and Daldal (1993) EMBO J 12: 1283–1292]. In this work, we have studied the kinetics of electron transfer between these secondary electron donors and the reaction center in intact cells of two mutants, MT-G4/S4 and MT-GS18 deleted in cytochrome c2 and in cytochrome c2 plus cytochrome bc1 complex, respectively. In the MT-G4/S4 mutant, only about one third of the primary electron donor is reduced by cytochrome cy in less than five ms. The remaining fraction is reduced in several seconds, although about 90% of the photoxidized cytochrome cy is reduced in less than 10 ms by the cytochrome bc1 complex. This implies that cytochrome cy is not in thermodynamic equilibrium with the large fraction of primary donors which are slowly reduced. As shown by energy transfer measurements, the reaction centers connected to cytochrome cy and the disconnected reaction centers are localized in the same membrane region. We propose that the movement of cyt cy is restricted to a small membrane domain which includes a single cytochrome bc1 complex. The kinetics of cytochrome cy photooxidation in the MT-G4/S4 mutant in the presence of myxothiazol presents a fast phase (t1/2 3 µs) followed by a slower phase (t1/2 20 µs). In the case of the double mutant MT-GS18, the kinetics of electron transfer between cytochrome cy and the reaction center is highly multiphasic and much slower than those observed for the MT-G4/S4 mutant. In particular, the amplitude of the fast phase is decreased by more than a factor 2 and the 20-µs phase is not observed. This implies an important structural role of the cytochrome bc1 complex in the interaction between reaction center and cytochrome cy, and their formation in supercomplex. The more problable stoichiometry of electron carriers in this supercomplex is 2 reaction centers, 2 cytochrome cy and 1 cytochrome bc1 complex. 相似文献
3.
Dumortier C Fitch J Meyer TE Cusanovich MA 《Archives of biochemistry and biophysics》2002,405(2):154-162
The Class I c-type cytochromes can bind exogenous ligands in the oxidized state, with the kinetics of ligand binding providing information on naturally occurring intramolecular dynamics. Typically, nitrogenous bases are used as ligands; however, it is less well known that 2-mercaptoethanol (BME), a commonly used cytochrome reducing agent, can form a complex with the heme. To better understand the cytochrome-mercaptan interaction, we have investigated the kinetics of binding of BME to wild type and mutants of Rhodobacter capsulatus cytochrome c(2) and to horse cytochrome c. Complex formation with the G95P mutant is apparent from the formation of a green color and a shift in the Soret peak to 418 nm from 410 nm upon addition of BME. Unlike horse cytochrome c and wild-type R. capsulatus cytochrome c(2), G95P permits the kinetics of formation of the BME-G95P complex to be measured since complex formation and reduction kinetics can be resolved. The affinity constant for the binding of BME to mutant G95P was strong ( approximately 1.5 x 10(5)M(-1)) and the kinetics of formation of the BME-G95P complex were found to undergo a change in rate-limiting step consistent with a concentration-independent protein rearrangement (68s(-1)) followed by second-order binding of BME ( approximately approximately 1.3 x 10(5)M(-1)s(-1)). The most remarkable characteristic of mutant G95P is the relatively large amount of high-spin species in equilibrium with the low- spin form, which can be estimated to be approximately 3% at pH 7. The BME binding kinetics, coupled with the kinetics of imidazole binding to G95P, allow us, for the first time, to specify all four rate constants describing the ligand binding reaction. Moreover, we can use the kinetic results to estimate the rate constants for ligand binding with the wild-type cytochrome c(2). This has also allowed us to quantify and more fully interpret cytochrome dynamics. 相似文献
4.
5.
Deng H 《Biochemical and biophysical research communications》2006,342(1):73-80
Cytochrome c and glutathione (GSH) are two important biomolecules that regulate many cellular processes. The reaction of cytochrome c with GSH involves radical oxygen species and exhibits significant complexity. In the present work, the reaction of cytochrome c with GSH in water was characterized using mass spectrometry. The results show for the first time that the reaction generates multiple products including apocytochrome c in oxidized and reduced forms, glutathionylated apocytochrome c, GSH-modified cytochrome c, and oxidized and hydroxylated species. The reaction is O(2) dependent and is rapid in water at neutral pH and 37 degrees C. The reaction involves the cleavage of thioether linkages between the heme and apocytochrome c. Evidence for the role of H(2)O(2) and other oxygen radicals in this reaction is also provided. 相似文献
6.
Cytochrome P450 2B4 is a microsomal protein with a multi-step reaction cycle similar to that observed in the majority of other cytochromes P450. The cytochrome P450 2B4-substrate complex is reduced from the ferric to the ferrous form by cytochrome P450 reductase. After binding oxygen, the oxyferrous protein accepts a second electron which is provided by either cytochrome P450 reductase or cytochrome b5. In both instances, product formation occurs. When the second electron is donated by cytochrome b5, catalysis (product formation) is ∼10- to 100-fold faster than in the presence of cytochrome P450 reductase. This allows less time for side product formation (hydrogen peroxide and superoxide) and improves by ∼15% the coupling of NADPH consumption to product formation. Cytochrome b5 has also been shown to compete with cytochrome P450 reductase for a binding site on the proximal surface of cytochrome P450 2B4. These two different effects of cytochrome b5 on cytochrome P450 2B4 reactivity can explain how cytochrome b5 is able to stimulate, inhibit, or have no effect on cytochrome P450 2B4 activity. At low molar ratios (<1) of cytochrome b5 to cytochrome P450 reductase, the more rapid catalysis results in enhanced substrate metabolism. In contrast, at high molar ratios (>1) of cytochrome b5 to cytochrome P450 reductase, cytochrome b5 inhibits activity by binding to the proximal surface of cytochrome P450 and preventing the reductase from reducing ferric cytochrome P450 to the ferrous protein, thereby aborting the catalytic reaction cycle. When the stimulatory and inhibitory effects of cytochrome b5 are equal, it will appear to have no effect on the enzymatic activity. It is hypothesized that cytochrome b5 stimulates catalysis by causing a conformational change in the active site, which allows the active oxidizing oxyferryl species of cytochrome P450 to be formed more rapidly than in the presence of reductase. 相似文献
7.
Nitrogen assimilation in Rhodobacter capsulatus has been shown to proceed via the coupled action of glutamine synthetase (GS) and glutamate synthase (GOGAT) with no measurable glutamate dehydrogenase (GDH) present. We have recently isolated a novel class of mutants of R. capsulatus strain B100 that lacks a detectable GOGAT activity but is able to grow at wild type rates under nitrogen-fixing conditions. While NH
4
+
-supported growth in the mutants was normal under anaerobic/photosynthetic conditions, the growth rate was decreased under aerobic conditions. Ammonium and methylammonium uptake experiments indicated that there was a clear difference in the ammonium assimilatory capabilities in these mutants under aerobic versus anaerobic growth. Regulation of expression of a nifH : : lacZ fusion in these mutants was not impaired. The possible existence of alternative ammonium assimilatory pathways is discussed. 相似文献
8.
The direct electron transfer of surface-confined horse heart cytochrome c (Cyt c) was achieved using COOH-terminated alkanethiolate-modified gold electrode. Later DNA was immobilized on the two-layer modified electrode. The quantitative determination of DNA was explored and the interaction between cytochrome c and DNA was studied. The binding site sizes were determined to be 15 bp per Cyt c molecule with double-stranded (ds) DNA and 30 nucleotides binding one Cyt c molecule with single-stranded (ss) DNA. At the dsDNA/Cyt c/MUA/Au electrode, the rate constant of oxidation electron transfer k(s,ox)=1.59x10(-3)cms-1 was obtained, at the ssDNA/Cyt c/MUA/Au electrode, the value was 2.43x10(-3)ms-1 when the scan rate was 1.0V/s. The different electrodes were characterized with electrochemical quartz crystal microbalance and atomic force microscope. 相似文献
9.
Lange C Hervás M De la Rosa MA 《Biochemical and biophysical research communications》2003,310(1):215-221
This work presents an improved stopped-flow protocol for the simultaneous measurement of thermodynamic and kinetic protein stability data from a single experiment, along with a formalism for the global analysis of the data. The method was applied to the comparison of the stabilities of cytochrome c(6) from Anabaena sp. PCC 7119 and one of its mutants (D72K). Compared to the wild type the mutant was found to have a significantly reduced thermodynamic (deltadeltaG(U0)=2.7 kJ mol(-1)) and kinetic stability, as well as a more pronounced shift in transition state structure upon destabilization. 相似文献
10.
The pH dependence of the Fe(III) reduction potential, E0′, for yeast cytochrome c peroxidase (yCcP) and three distal pocket mutants, CcP(H52L), CcP(H52Q), and CcP(R48L/W51L/H52L), has been determined between pH 4 and 8. E0′ values at pH 7.0 for the yCcP, CcP(H52L), CcP(H52Q), and CcP(R48L/W51L/H52L) are − 189, − 170, − 224, and − 146 mV, respectively. A heme-linked ionization in the reduced enzyme affects the reduction potential for yCcP and all three mutants. Apparent pKA values for the heme-linked ionization are 7.5 ± 0.2, 6.5 ± 0.3, 6.4 ± 0.2, and 7.0 ± 0.3 for yCcP and the H52L, H52Q, and R48L/W51L/H52L mutants, respectively. A cooperative, two-proton ionization causing a spectroscopically-detectable transition was observed in the ferrous states of yCcP, CcP(H52L) and CcP(H52Q), with apparent pKA values of 7.7 ± 0.2, 7.4 ± 0.1 and 7.8 ± 0.1, respectively. These data indicate that: (1) the distal histidine in CcP is not the site of proton binding upon reduction of the ferric CcP, (2) the distal histidine is not one of the two groups involved in the cooperative, two-proton ionization observed in ferrous CcP, and (3) the proton-binding site is not involved in the cooperative, two-proton ionization observed in the reduced enzyme. 相似文献
11.
The physiological regulation of mitochondrial respiration by NO has been reported to result from the reversible binding of NO to the two-electron reduced binuclear center (Fe(2+)(a3)-Cu(1+)(B)) of cytochrome c oxidase (CcO). Although the role of CcO and its derived catalytic intermediates in the catabolism of NO has been documented, little has been established for the enzyme in its fully oxidized state (Fe(3+)(a3)-Cu(2+)(B)). We report: (1) CcO, in its fully oxidized state, represents the major component of the mitochondrial electron transport chain for NO consumption as controlled by the binding of NO to its binuclear center. Phospholipid enhances NO consumption by fully oxidized CcO, whereas the consumption of NO is slowed down by membrane structure and membrane potential when CcO is embedded in the phospholipid bilayer. (2) In the presence of H(2)O(2), CcO was shown to serve as a mitochondria-derived NO peroxidase. A CcO-derived protein radical intermediate was induced and involved in the modulation of NO catabolism. 相似文献
12.
The cbb3-type cytochrome c oxidases as members of the heme-copper oxidase superfamily are involved in microaerobic respiration in both pathogenic and non-pathogenic proteobacteria. The biogenesis of these multisubunit enzymes, encoded by the ccoNOQP operon, depends on the ccoGHIS gene products, which are proposed to be specifically required for co-factor insertion and maturation of cbb3-type cytochrome c oxidases. Here, the assembly of the cbb3-type cytochrome c oxidase from the facultative photosynthetic model organism Rhodobacter capsulatus was investigated using blue-native polyacrylamide gel electrophoresis. This process involves the formation of a stable but inactive 210 kDa sub-complex consisting of the subunits CcoNOQ and the assembly proteins CcoH and CcoS. By recruiting monomeric CcoP, this sub-complex is converted into an active 230 kDa CcoNOQP complex. Formation of these complexes and the stability of the monomeric CcoP are impaired drastically upon deletion of ccoGHIS. In a ccoI deletion strain, the 230 kDa complex was absent, although monomeric CcoP was still detectable. In contrast, neither of the complexes nor the monomeric CcoP was found in a ccoH deletion strain. In the absence of CcoS, the 230 kDa complex was assembled. However, it exhibited no enzymatic activity, suggesting that CcoS might be involved in a late step of biogenesis. Based on these data, we propose that CcoN, CcoO and CcoQ assemble first into an inactive 210 kDa sub-complex, which is stabilized via its interactions with CcoH and CcoS. Binding of CcoP, and probably subsequent dissociation of CcoH and CcoS, then generates the active 230 kDa complex. The insertion of the heme cofactors into the c-type cytochromes CcoP and CcoO precedes sub-complex formation, while the cofactor insertion into CcoN could occur either before or after the 210 kDa sub-complex formation during the assembly of the cbb3-type cytochrome c oxidase. 相似文献
13.
Bcl-x(S), a pro-apoptotic member of the Bcl-2 protein family, is localized in the mitochondrial outer membrane and induces caspase-dependent and nerve growth factor (NGF)-inhibitable apoptosis in PC12 cells. The mechanism of action of Bcl-x(S) and how NGF inhibits this death are not fully understood. It is still unknown whether Bcl-x(S) induces mitochondrial cytochrome c release, and which apoptotic step NGF inhibits. We show that Bcl-x(S) induces cytochrome c release and caspase-3 activation in several cell types, and that in PC12 cells, these events are inhibited by NGF treatment. The survival effect of NGF was inhibited by inhibitors of protein kinase C (PKC), phosphatidylinositol-3-kinase (PI 3-kinase), and the mitogen-activated protein kinase kinase (MEK) inhibitors GF109203X, LY294002, and U0126. These findings show that cytochrome c release and caspase-3 activation participate in Bcl-x(S)-induced apoptosis, and that NGF inhibits Bcl-x(S)-induced apoptosis at the mitochondrial level via the PKC, PI 3-kinase, and MEK signaling pathways. 相似文献
14.
Solís C Celis H Romero I Isaac-Olivé K Andrade E Eslava-Rosas F Monroy-Rosales JH Cuevas-Moreno R 《Journal of microbiological methods》2011,84(2):272-277
Inorganic pyrophosphatases are divided in two families, which differ both in structure and mechanism. All of them incorporate in its structure divalent metal cations. In 2003, it was reported for the first time that Rhodobacter capsulatus cytoplasmic pyrophosphatase belongs to family II. It is expected then, that this enzyme contains metal elements in its structure; however, this characterization has not been carried out yet. A fine application of accelerators is the use of proton beams to induce X-ray emission (PIXE) for analyzing the composition of biological macromolecules. The purpose of this work is to complement R. capsulatus cytoplasmic pyrophosphatase characterization by determining the presence of metal elements in its structure. Three different strategies were used: PAGE-PIXE, PAGE-Digestion-PIXE, and Dialysis-PIXE and when metals were found the metal/enzyme ratio was calculated. Only Cobalt was found to be associated to the enzyme chemical structure in a ratio 3 Co/enzyme. 相似文献
15.
Stoyanovsky DA Vlasova II Belikova NA Kapralov A Tyurin V Greenberger JS Kagan VE 《FEBS letters》2008,582(5):725-728
In mitochondrial apoptosis, the formation of cytochrome c-cardiolipin complex ([CL-cyt c]) with peroxidase properties is an early event in the cascade of reactions that leads to cell death. Herein, we report the synthesis of a new prodrug, (2-hydroxyamino-vinyl)-triphenyl-phosphonium (HVTP), which compartmentalizes exclusively into mitochondria, undergoes a [CL-cyt c]-catalyzed bioactivation to nitric oxide (NO), inhibits peroxidase activity, and protects cells from apoptosis. 相似文献
16.
Rodríguez-Roldán V García-Heredia JM Navarro JA Hervás M De la Cerda B Molina-Heredia FP De la Rosa MA 《Biochemical and biophysical research communications》2006,346(3):1108-1113
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. 相似文献
17.
The production of biomass, polysaccharide storage material and H2 from malate was studied in the wild-type and mutants RdcI, RdcII and RdcI/cII of Rhodobacter capsulatus. The mutants are defective in either copy I, copy II or both copies of the nitrogenase genes nifA and nifB. Stationary phase levels of biomass, polysaccharide and H2 were determined in phototrophic batch cultures grown with 30 mM of d,l-malate and either 2, 5, or 8 mM of ammonium or 7 mM of glutamate. Calculation of the amounts of malate converted into the
three products revealed that, at 8 mM of ammonium and 7 mM of glutamate, malate consumption and product formation were balanced.
But with decreasing ammonium concentrations malate not converted into biomass was utilized with decreasing efficiency in polysaccharide
and H2 formation. This suggests formation of unknown products at the lower ammonium concentrations. Under conditions of optimal
N supply, 80% of the malate not used for biomass production was converted by the wild-type and strain RdcII to H2 and CO2. Mutant RdcI exhibited slightly decreased H2 production. The double mutant did not evolve H2 but accumulated increased amounts of polysaccharide. However, the amounts of polysaccharide were lower than should be expected
if all of the spare malate, not utilized by the double mutant for H2 production, was converted into storage material. This and incomplete conversion of malate into known products at low ammonium
supplies suggests that polysaccharide accumulation does not compete with the process of H2 formation for malate. 相似文献
18.
A promoted electron transfer of an antitumor drug, mitoxantrone (MTX), intercalating into DNA duplex was successfully obtained upon addition of cytochromes c (cyt. c) in NaAc-HAc buffer solution (pH 4.5). The experimental results suggested that co-existence of MTX and cyt. c in the DNA helix is an important factor for accelerated electron transfer of MTX, where the promoter, cyt. c, operated smoothly through the DNA bridge. The UV/Vis spectroscopic experiments further confirmed the interaction process. Furthermore, a possible mechanism of such reaction was also discussed in this paper. 相似文献
19.
Two conserved non-canonical histidines are essential for activity of the cbb
3-type oxidase in Rhodobacter capsulatus 总被引:1,自引:0,他引:1
Cytochrome cbb
3 oxidase, a member of the heme–copper oxidase superfamily, catalyses the reduction of oxygen to water and generates a proton
gradient. Cytochrome c oxidases are characterized by a catalytic subunit (subunit I) containing two hemes and one copper ion ligated by six invariant
histidine residues, which are diagnostic of heme–copper oxidases in all type of the heme–copper oxidase superfamily. Alignments
of the amino acid sequences of subunit I (FixN or CcoN) of the cbb
3-type oxidases show that catalytic subunit also contains six non-canonical histidine residues that are conserved in all CcoN
subunits of the cbb
3 oxidase, but not the catalytic subunits of other members of heme–copper oxidases superfamily. The function of these six CcoN-specific
conserved histidines of cbb
3-type oxidase in R. capsulatus is unknown. To analyze the contribution of the two invariant histidines of CcoN, H300 and H394, in activity and assembly
of the Rhodobacter capsulatus
cbb
3-type oxidase, they were substituted for valine and alanine, respectively by site-directed mutagenesis. H300V and H394A mutations
were analyzed with respect to their activity and assembly. It was found that H394A mutation led to a defect in the assembly
of both CcoP and CcoO in the membrane, which results in almost complete loss of activity and that although the H300V mutant
is normally assembled in the membrane and retain their stability, its catalytic activity is significantly reduced when compared
with wild-type oxidase. 相似文献
20.
Challenge of Rhodobacter capsulatus cells with the superoxide propagator methyl viologen resulted in the induction of a diaphorase activity identified as a member of the ferredoxin (flavodoxin)-(reduced) nicotinamide adenine dinucleotide phosphate (NADP(H)) reductase (FPR) family by N-terminal sequencing. The gene coding for Rhodobacter FPR was cloned and expressed in Escherichia coli. Both native and recombinant forms of the enzyme were purified to homogeneity rendering monomeric products of approximately 30 kDa with essentially the same spectroscopic and kinetic properties. They were able to bind and reduce Rhodobacter flavodoxin (NifF) and to mediate typical FPR activities such as the NADPH-driven diaphorase and cytochrome c reductase. 相似文献