[Peculiarities of the Structural-Functional State of the Cytochrome Part of Liver Mitochondrial Respiratory Chain under Conditions of Acetaminophen-induced Hepatitis against the Background of Alimentary Deprivation of Protein].

Activity of the key enzyme of the cytochrome part of the respiratory chain--cytochrome oxidase, quantitative redistribution of mitochondrial cytochromes b, c1, c and aa3, activity of the key enzymes of cytochromes' heme metabolism--delta-aminolevulinate synthase and heme oxygenase under conditions of acetaminophen-induced hepatitis against the background of alimentary deprivation of protein were studied. It was found out, that under conditions of acetaminophen-induced hepatitis against the background of alimentary deprivation of protein, an inhibition of cytochrome oxidase activity and a decrease in the quantitative content of mitochondrial cytochromes against the background of the increase in the delta-aminolevulinate synthase and heme oxygenase activity are observed. In animals with toxic liver injury, maintained under conditions of alimentary deprivation of protein, a progressive decrease in the quantitative content of mitochondrial cytochromes b, c1, c and aa3 against the background. of the increase in heme oxygenase activity and preservation of delta-aminolevulinate synthase activity on the control level is identified. The conclusion was made, that alimentary deprivation of protein is a critical factor for the development of the disturbances of structural-functional integrity of the cytochromic part of the respiratory chain. The identified changes may be considered as one of the possible mechanisms of energy biotransformation system disturbances under conditions of alimentary deprivation of protein.

Correlations between the Electronic Properties of Shewanella oneidensis Cytochrome c Nitrite Reductase (ccNiR) and Its Structure: Effects of Heme Oxidation State and Active Site Ligation.

The electrochemical properties of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), a homodimer that contains five hemes per protomer, were investigated by UV-visible and electron paramagnetic resonance (EPR) spectropotentiometries. Global analysis of the UV-vis spectropotentiometric results yielded highly reproducible values for the heme midpoint potentials. These midpoint potential values were then assigned to specific hemes in each protomer (as defined in previous X-ray diffraction studies) by comparing the EPR and UV-vis spectropotentiometric results, taking advantage of the high sensitivity of EPR spectra to the structural microenvironment of paramagnetic centers. Addition of the strong-field ligand cyanide led to a 70 mV positive shift of the active site's midpoint potential, as the cyanide bound to the initially five-coordinate high-spin heme and triggered a high-spin to low-spin transition. With cyanide present, three of the remaining hemes gave rise to distinctive and readily assignable EPR spectral changes upon reduction, while a fourth was EPR-silent. At high applied potentials, interpretation of the EPR spectra in the absence of cyanide was complicated by a magnetic interaction that appears to involve three of five hemes in each protomer. At lower applied potentials, the spectra recorded in the presence and absence of cyanide were similar, which aided global assignment of the signals. The midpoint potential of the EPR-silent heme could be assigned by default, but the assignment was also confirmed by UV-vis spectropotentiometric analysis of the H268M mutant of ccNiR, in which one of the EPR-silent heme's histidine axial ligands was replaced with a methionine.

CYC1 silencing sensitizes osteosarcoma cells to TRAIL-induced apoptosis.

AIMS: Osteosarcoma (OS) is an aggressive bone malignancy with poor prognosis. Many OS cells are resistant to apoptotic induction by tumor necrosis factor-related apoptosis inducing ligand (TRAIL). In our previous study, we found that the serum level of cytochrome c1 (CYC1) is significantly higher in OS patients than in healthy subjects. Our aim was to investigate the effects of CYC1 silencing on TRAIL-induced apoptosis in human OS in vitro and in vivo along with the underlying mechanisms. METHODS: First, we determined the expression of CYC1 in human OS tumors and cell lines versus normal adjacent tissues and cell line. We then studied the effects of CYC1 silencing alone or in combination with TRAIL on OS cell growth and apoptosis in vitro and OS tumorigenesis in vivo. RESULTS: We found that CYC1 is overexpressed in human OS tissues and cell lines. CYC1 silencing by shRNA transfection inhibits proliferation, slightly induces apoptosis in human OS cells in vitro, and suppresses human OS tumor growth in a mouse xenograft model in vivo. Additionally, CYC1 silencing sensitizes OS to TRAIL-induced apoptosis in vitro and in vivo. Our results also showed that CYC1 silencing significantly reduces complex III activity and potentiates TRAIL-induced cytochrome c release and caspase-9 activation in OS cells, suggesting that CYC1 silencing acts via the mitochondria-dependent apoptotic pathway. CONCLUSION: Taken together, our results provide evidence that CYC1 plays an important role in OS tumorigenesis, and modulation of CYC1 may be an effective strategy to potentiate OS to apoptotic induction by TRAIL.

Combining Inhibitor Resistance-conferring Mutations in Cytochrome b Creates Conditional Synthetic Lethality in Saccharomyces cerevisiae.

The mitochondrial cytochrome bc(1) complex is an essential respiratory enzyme in oxygen-utilizing eukaryotic cells. Its core subunit, cytochrome b, contains two sites, center P and center N, that participate in the electron transfer activity of the bc(1) complex and that can be blocked by specific inhibitors. In yeast, there are various point mutations that confer inhibitor resistance at center P or center N. However, there are no yeast strains in which the bc(1) complex is resistant to both center P and center N inhibitors. We attempted to create such strains by crossing yeast strains with inhibitor-resistant mutations at center P with yeast strains with inhibitor-resistant mutations at center N. Characterization of yeast colonies emerging from the cross revealed that there were multiple colonies resistant against either inhibitor alone but that the mutational changes were ineffective when combined and when the yeast were grown in the presence of both inhibitors. Inhibitor titrations of bc(1) complex activities in mitochondrial membranes from the various yeast mutants showed that a mutation that confers resistance to an inhibitor at center P, when combined with a mutation that confers resistance to an inhibitor at center N, eliminates or markedly decreases the resistance conferred by the center N mutation. These results indicate that there is a pathway for structural communication between the two active sites of cytochrome b and open new possibilities for the utilization of center N as a potential drug target.

In situ kinetics of cytochromes c1 and c2.

In Rhodobacter sphaeroides chromatophores, cytochromes (cyt) c(1) and c(2) have closely overlapping spectra, and their spectral deconvolution provides a challenging task. As a result, analyses of the kinetics of different cytochrome components of the bc(1) complex in purple bacteria usually report only the sum cyt c(1) + cyt c(2) kinetics. Here we used newly determined difference spectra of individual components to resolve the kinetics of cyt c(1) and c(2) in situ via a least-squares (LS) deconvolution. We found that the kinetics of cyt c(1) and c(2) are significantly different from those measured using the traditional difference wavelength (DW) approach, based on the difference in the absorbance at two different wavelengths specific for each component. In particular, with the wavelength pairs previously recommended, differences in instrumental calibration led to kinetics of flash-induced cyt c(1) oxidation measured with the DW method which were faster than those determined by the LS method (half-time of approximately 120 micros vs half-time of approximately 235 micros, in the presence of antimycin). In addition, the LS approach revealed a delay of approximately 50 micros in the kinetics of cyt c(1) oxidation, which was masked when the DW approach was used. We attribute this delay to all processes leading to the oxidation of cyt c(1) after light activation of the photosynthetic reaction center, especially the dissociation of cyt c(2) from the reaction center. We also found that kinetics of both cyt c(1) and c(2) measured by the DW approach were significantly distorted at times longer than 1 ms, due to spectral contamination from changes in the b hemes. The successful spectral deconvolution of cyt c(1) and c(2), and inclusion of both cytochromes in the kinetic analysis, significantly increase the data available for mechanistic understanding of bc(1) turnover in situ.

Inhibiting Escherichia coli cytochrome c nitrite reductase: voltammetry reveals an enzyme equipped for action despite the chemical challenges it may face in vivo.

Escherichia coli cytochrome c nitrite reductase is one of a large family of homologous enzymes that are particularly prevalent in pathogenic enterobacteria. The enzymes are periplasmic and in vivo may find themselves challenged by molecules that could enhance or compromise their performance. In the present study, we describe protein film voltammetry in which the activity of E. coli cytochrome c nitrite reductase is challenged by the presence of a number of small molecules. These results are discussed in light of the environment(s) that the enzyme may face before and after colonization of a human host.

Resolving complexity in the interactions of redox enzymes and their inhibitors: contrasting mechanisms for the inhibition of a cytochrome c nitrite reductase revealed by protein film voltammetry.

Cytochrome c nitrite reductase is a dimeric decaheme-containing enzyme that catalyzes the reduction of nitrite to ammonium. The contrasting effects of two inhibitors on the activity of this enzyme have been revealed, and defined, by protein film voltammetry (PFV). Azide inhibition is rapid and reversible. Variation of the catalytic current magnitude describes mixed inhibition in which azide binds to the Michaelis complex (approximately 40 mM) with a lower affinity than to the enzyme alone (approximately 15 mM) and leads to complete inhibition of enzyme activity. The position of the catalytic wave reports tighter binding of azide when the active site is oxidized (approximately 39 microM) than when it is reduced. By contrast, binding and release of cyanide are sluggish. The higher affinity of cyanide for reduced versus oxidized forms of nitrite reductase is immediately revealed, as is the presence of two sites for cyanide binding and inhibition of the enzyme. Formation of the monocyano complex by reduction of the enzyme followed by a "rapid" scan to high potentials captures the activity-potential profile of this enzyme form and shows it to be distinct from that of the uninhibited enzyme. The biscyano complex is inactive. These studies demonstrate the complexity that can be associated with inhibitor binding to redox enzymes and illustrate how PFV readily captures and deconvolves this complexity through its impact on the catalytic properties of the enzyme.

Redox-triggered events in cytochrome c nitrite reductase.

Escherichia coli cytochrome c nitrite reductase is a homodimeric enzyme whose 10 heme centres range in reduction potential from ca. -30 to -320 mV. Protein film voltammetry (PFV) was performed to assess how the reactivity of the enzyme towards a number of small molecules was influenced by heme oxidation state. The experimental approach provided a high-resolution description of activity across the electrochemical potential domain by virtue of the fact that the enzyme sample was under the precise potential control of an electrode at all times. The current potential profiles displayed by nitrite reductase revealed that heme oxidation state has a profound, and often unanticipated, effect on the interactions with substrate molecules, nitrite and hydroxylamine, as well as the inhibitor, cyanide. Thus, PFV provides a powerful route to define redox-triggered events in this complex multi-centred redox enzyme.

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.

Effect of famoxadone on photoinduced electron transfer between the iron-sulfur center and cytochrome c1 in the cytochrome bc1 complex.

Famoxadone is a new cytochrome bc(1) Q(o) site inhibitor that immobilizes the iron-sulfur protein (ISP) in the b conformation. The effects of famoxadone on electron transfer between the iron-sulfur center (2Fe-2S) and cyt c(1) were studied using a ruthenium dimer to photoinitiate the reaction. The rate constant for electron transfer in the forward direction from 2Fe-2S to cyt c(1) was found to be 16,000 s(-1) in bovine cyt bc(1). Binding famoxadone decreased this rate constant to 1,480 s(-1), consistent with a decrease in mobility of the ISP. Reverse electron transfer from cyt c(1) to 2Fe-2S was found to be biphasic in bovine cyt bc(1) with rate constants of 90,000 and 7,300 s(-1). In the presence of famoxadone, reverse electron transfer was monophasic with a rate constant of 1,420 s(-1). It appears that the rate constants for the release of the oxidized and reduced ISP from the b conformation are the same in the presence of famoxadone. The effects of famoxadone binding on electron transfer were also studied in a series of Rhodobacter sphaeroides cyt bc(1) mutants involving residues at the interface between the Rieske protein and cyt c(1) and/or cyt b.

2,6-Dichloro-phenol indophenol prevents switch-over of electrons between the cyanide-sensitive and -insensitive pathway of the mitochondrial electron transport chain in the presence of inhibitors.

To evolve a simple oxygen electrode-based method to estimate alternative respiration, one needs to develop a procedure to prevent switch-over of electrons to either pathway upon inhibition by cyanide or salicylhydroxamic acid. It was hypothesized that the inclusion of appropriate electron acceptor, possessing redox potential close to one of the electron transport carriers in between ubiquinone (branch point) and cytochrome a-a3, should be able to stop switch-over of electrons to either pathway by working as an electron sink. To test the hypothesis, 2,6-dichloro-phenol indophenol (DCPIP; redox potential +0.217 V), an artificial electron acceptor having a redox potential quite similar to the site near cytochrome c1 (redox potential +0.22 V) on the cyanide-sensitive pathway, was used with isolated mitochondria and leaf discs in the absence and presence of inhibitors (potassium cyanide, antimycin A, and salicylhydroxamic acid). Polarographic data confirmed electron acceptance by DCPIP only from the inhibited (by cyanide or salicylhydroxamic acid) mitochondrial electron transport chain, hence preventing switch-over of electrons between the cyanide-sensitive and cyanide-insensitive pathway of respiration. Results with antimycin A and reduction status of DCPIP further confirmed electron acceptance by DCPIP from the mitochondrial electron transport chain. Possible implications of the results have been discussed.

The oxidation-reduction kinetics of cytochromes b, c1 and c in initially fully reduced mitochondrial membranes are in agreement with the Q-cycle hypothesis.

Stopped-flow experiments were performed to distinguish between two hypotheses, the Q-cycle and the SQ-cycle, each describing the pathway of electron transfer in the QH2:cytochrome c oxidoreductases. It was observed that, when mitochondrial membranes from the yeast Saccharomyces cerevisiae were poised at a low redox potential with appropriate amounts of sodium dithionite to completely reduce cytochrome b, the kinetics of oxidation of cytochrome b showed a lag period of maximally 100 ms. Under the same experimental conditions, the oxidation-reduction kinetics of cytochromes c + c1 showed transient behaviour. These results do not support the presence of a mobile species of semiquinone in the QH2:cytochrome c oxidoreductases, as envisaged in the SQ-cycle, but are consistent with a Q-cycle mechanism in which the two quinone-binding domains do not exchange electrons directly on the timescale of turnover of the enzyme.

Inhibitors of the mitochondrial cytochrome b-c1 complex inhibit the cyanide-insensitive respiration of Trypanosoma brucei.

The cyanide-insensitive respiration of bloodstream trypomastigote forms of Trypanosoma brucei (75 +/- 8 nmol O2 min-1(mg protein)-1) is completely inhibited by the mitochondrial ubiquinone-like inhibitors 2-hydroxy-3-undecyl-1,4-naphthoquinone (UHNQ) and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). The Ki values for UHDBT (30 nM) and UHNQ (2 microM) are much lower than the reported Ki for salicylhydroxamic acid (SHAM) (5 microM), a widely used inhibitor of the cyanide-insensitive oxidase. UHNQ also stimulated the glycerol-3-phosphate-dependent reduction of phenazine methosulfate, demonstrating that the site of UHNQ inhibition is on the terminal oxidase of the cyanide-insensitive respiration of T. brucei. These results suggest that a ubiquinone-like compound may act as an electron carrier between the two enzymatic components of the cyanide-insensitive glycerol-3-phosphate oxidase.

Effect of 2,4-dinitrofluorobenzene on the enzymatic properties of the b-c1 complex isolated from beef heart mitochondria.

A study is presented on the effect of 2,4-dinitrofluorobenzene (DFNB) on the enzymatic properties of mitochondrial b-c1 complex. The chemical modification by DNFB strongly inhibits the reductase activity of the complex, this being accompanied by labelling by [3H]DNFB of core protein I, the apoprotein of b cytochromes and the 12 kDa subunit. Chemical modification by DNFB appears to alter, in particular, the domain of heme b-562.