Understanding the chemistry of the artificial electron acceptors PES, PMS, DCPIP and Wurster's Blue in methanol dehydrogenase assays.

Methanol dehydrogenases (MDH) have recently taken the spotlight with the discovery that a large portion of these enzymes in nature utilize lanthanides in their active sites. The kinetic parameters of these enzymes are determined with a spectrophotometric assay first described by Anthony and Zatman 55 years ago. This artificial assay uses alkylated phenazines, such as phenazine ethosulfate (PES) or phenazine methosulfate (PMS), as primary electron acceptors (EAs) and the electron transfer is further coupled to a dye. However, many groups have reported problems concerning the bleaching of the assay mixture in the absence of MDH and the reproducibility of those assays. Hence, the comparison of kinetic data among MDH enzymes of different species is often cumbersome. Using mass spectrometry, UV-Vis and electron paramagnetic resonance (EPR) spectroscopy, we show that the side reactions of the assay mixture are mainly due to the degradation of assay components. Light-induced demethylation (yielding formaldehyde and phenazine in the case of PMS) or oxidation of PES or PMS as well as a reaction with assay components (ammonia, cyanide) can occur. We suggest here a protocol to avoid these side reactions. Further, we describe a modified synthesis protocol for obtaining the alternative electron acceptor, Wurster's blue (WB), which serves both as EA and dye. The investigation of two lanthanide-dependent methanol dehydrogenases from Methylorubrum extorquens AM1 and Methylacidiphilum fumariolicum SolV with WB, along with handling recommendations, is presented. Lanthanide-dependent methanol dehydrogenases. Understanding the chemistry of artificial electron acceptors and redox dyes can yield more reproducible results.

Effect of artificial redox mediators on the photoinduced oxygen reduction by photosystem I complexes.

The peculiarities of interaction of cyanobacterial photosystem I with redox mediators 2,6-dichlorophenolindophenol (DCPIP) and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) were investigated. The higher donor efficiency of the reduced DCPIP form was demonstrated. The oxidized form of DCPIP was shown to be an efficient electron acceptor for terminal iron-sulfur cluster of photosystem I. Likewise methyl viologen, after one-electron reduction, DCPIP transfers an electron to the molecular oxygen. These results were discussed in terms of influence of these interactions on photosystem I reactions with the molecular oxygen and natural electron acceptors.

Inhibition of reversed electron transfer and proton transport in the beef heart cytochrome bc1 complex by chemical modification.

Chemical modification of the bovine heart cytochrome bc1 complex with N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) has been reported to inhibit the proton pumping activity without affecting the rate of electron transfer to ferricytochrome c. This study aims to examine the effect of EEDQ on energy-linked reversed electron transfer in the bc1 complex reconstituted into potassium-loaded phospholipid vesicles. Generation of a valinomycin-mediated potassium-diffusion potential induced the reduction of cytochrome b in the reconstituted bc1 complex in the presence of sodium ascorbate. The time course of the cytochrome b reduction was well correlated with that of the absorbance change of safranine, an optical probe for measuring membrane potential. Treatment of the bc1 complex with EEDQ caused a decrease in the potential-induced reduction of cytochrome b as well as in the proton translocation activity. But a significant loss in the ubiquinol-cytochrome c reducing activity was not observed in the EEDQ-treated bc1 complex. The time- and concentration-dependent effect of EEDQ on the reversed electron transfer was well correlated with that of the proton translocation activity of the bc1 complex. These findings strongly support the idea that the potential-induced reversal of electron transfer is coupled to the reverse flow of protons in the cytochrome bc1 complex.

Effect of dehydroepiandrosterone (DHEA) treatment on oxidative energy metabolism in rat liver and brain mitochondria. A dose-response study.

OBJECTIVES: Effects of treatment with dehydroepiandrosterone (DHEA) on oxidative energy metabolism in rat liver and brain mitochondria were examined. DESIGN AND METHODS: Young adult rats were administered DHEA (0.1, 0.2, 1.0 or 2.0 mg/kg body weight) by subcutaneous route for 7 consecutive days. RESULTS: DHEA treatment resulted in general, in stimulation of state 3 respiration rates without having any uncoupling effect on ADP/O ratios. The stimulation of state 3 respiration rate for a given substrate was dose dependent in a tissue-specific manner. Parallel increases in the contents of cytochromes aa(3) and b were also noted. DHEA treatment stimulated the glutamate dehydrogenase (GDH) and succinate DCIP reductase (SDR) activities. Under the treatment conditions, mitochondrial ATPase activity was also stimulated. CONCLUSIONS: Treatment with DHEA significantly stimulated oxidative energy metabolism in liver and brain mitochondria.

Cytochrome c association with the inner mitochondrial membrane is impaired in the CNS of G93A-SOD1 mice.

A "gain-of-function" toxic property of mutant Cu-Zn superoxide dismutase 1 (SOD1) is involved in the pathogenesis of some familial cases of amyotrophic lateral sclerosis (ALS). Expression of a mutant form of the human SOD1 gene in mice causes a degeneration of motor neurons, leading to progressive muscle weakness and hindlimb paralysis. Transgenic mice overexpressing a mutant human SOD1 gene (G93A-SOD1) were used to examine the mitochondrial involvement in familial ALS. We observed a decrease in mitochondrial respiration in brain and spinal cord of the G93A-SOD1 mice. This decrease was significant only at the last step of the respiratory chain (complex IV), and it was not observed in transgenic wild-type SOD1 and nontransgenic mice. Interestingly, this decrease was evident even at a very early age in mice, long before any clinical symptoms arose. The effect seemed to be CNS specific, because no decrease was observed in liver mitochondria. Differences in complex IV respiration between brain mitochondria of G93A-SOD1 and control mice were abolished when reduced cytochrome c was used as an electron donor, pinpointing the defect to cytochrome c. Submitochondrial studies showed that cytochrome c in the brain of G93A-SOD1 mice had a reduced association with the inner mitochondrial membrane (IMM). Brain mitochondrial lipids, including cardiolipin, had increased peroxidation in G93A-SOD1 mice. These results suggest a mechanism by which mutant SOD1 can disrupt the association of cytochrome c with the IMM, thereby priming an apoptotic program.

NADH oxidation by quinone electron acceptors.

The rate constants of NADH oxidation by quinones are increased with the oxidation potential increase: log kox (M-1 X s-1) = -0.25 + 12.2 E0(7) (V) for o-quinones and log kox (M-1 X s-1) = -3.06 + 13.5 E0(7) (V) for p-quinones (pH 7.0, 25 degrees C). It is assumed that the oxidation proceeds via the hydride-ion transfer. The rate constants of NADH oxidation by single-electron quinone acceptors are also increased with the oxidizer potential increase; log kox (M-1 X s-1) = -0.64 + 9.34 E0(7) (V) and correlate with the constants of NADH oxidation by quinone radicals obtained earlier (Grodkowski, J., Neta, P., Carlson, B.W. and Miller, L. (1983) J. Phys. Chem. 87, 3135-3138). Single-electron transfer is the limiting stage of the process.

Kinetics of cytochrome c and TMPD oxidation by cytochrome c oxidase from the thermophilic bacterium, PS3.

Cytochrome caa3 (cytochrome oxidase) from the thermophilic bacterium PS3 can exhibit full catalytic activity in the presence of ascorbate and TMPD or other electron donors and in the absence of added soluble c-type cytochromes. It appears to possess only a low-affinity and not a high-affinity site for the soluble cytochromes. Proteoliposomal cytochrome caa3 develops an effective membrane potential in the presence of ascorbate and TMPD or PMS, in the absence of added soluble cytochrome c. Reduction of the a3 centre is blocked in the presence of cyanide. During reductive titrations of the cyanide-inhibited enzyme, electrons initially equilibrate among three centres, the c haem, the a haem and one of the associated Cu atoms. During steady-state turnover, electrons probably enter the complex via the bound c haem; the a haem and perhaps an associated CuA atom are reduced next. It is concluded that, despite its size and hydrophobic association with the aa3 complex, the haem c-containing subunit can behave in an analogous way to that of mammalian cytochrome c, bound at the high-affinity site of the eucaryotic enzyme.

The respiratory chain of Azotobacter vinelandii. III. The effect of cyanide in the presence of substrates.

(1) Cyanide (100 microM) causes a rapid disappearance of the band (648 nm) of oxidized cytochrome d in particles of Azotobacter vinelandii oxidizing NADH. The rate of disappearance of the band can be related to the rate of inhibition of the oxygen consumption. (2) The kinetics of the disappearance of the 648-nm band of cytochrome d with excess cyanide in the presence of substrates deviate from first-order, indicating that at least two conformations of the enzyme are involved. (3) The rate of binding of cyanide to cytohrrome d increases the larger the rate of turnover of the oxidase. From this it is concluded that cyanide binds preferentially to the enzymically active oxidized conformation of cytochrome d. (4) The instantaneous inhibition of the oxidation of ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) by cyanide is related neither to the binding of cyanide to cytochrome d as determined spectrophotometrically, nor to the rate of inhibition of the oxidation of NADH. This indicates that different oxidases are involved in the oxidation of NADH and of ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), in line with the conclusion of Jones and Redfearn (1967) Biochim. Biophys. Acta 143, 340-353. (5) From experiments in which the change in redox states of cytochrome b1 and c-551 were related to the rate of binding of cyanide to cytochrome d, it is concluded that the cytochromes b1 and d are located in the main pathway to oxygen, whereas cytochrome c-551 functions in the branch via cytochrome o. (6) After prolonged incubation of particles with cyanide in the presence of NADH a residual activity (5%) was found in which a b-type cytochrome is involved. This shows the existence of a third but minor pathway to oxygen.

Isolation and purification of the cytochrome oxidase of Azotobacter vinelandii.

A membrane-bound cytochrome oxidase for Azobacter vinelandii was purified 20-fold using a detergent-solubilization procedure. Activity was monitored using as ascorbate-TMPD oxidation assay. The oxidase was 'solubilized' from a sonic-type electron-transport particle (R3 fraction) using Triton X-100 and deoxycholate. Low detergent concentrations first solubilized the flavoprotein oxidoreductases, then higher concentrations of Triton X-100 and KCl solubilized the oxidase, which was precipitated at 27-70% (NH4)2SO4. The highly purified cytochrome oxidase has a V of 60-78 microgatom O consumed/min per mg protein. TMPD oxidation by the purified enzyme was inhibited by CO, KCN, NaN3 and NH2OH; NaNO2 (but not NaNO3) also had a potent inhibitory effect. Spectral analyses revealed two major hemoproteins, the c-type cytochrome c4 and cytochrome o; cytochromes a1 and d were not detected. The Azotobacter cytochrome oxidase is an integrated cytochrome c4-o complex, TMPD-dependent cytochrome oxidase activity being highest in preparations having a high c-type cytochrome content. This TMPD-dependent cytochrome oxidase serves as a major oxygen-activation site for the A. vinelandii respiratory chain. It appears functionally analogous to cytochrome a+a3 oxidase of mammalian mitochondria.

On the stoichiometry between uncouplers of oxidative phosphorylation and respiratory chains. The catalytic action of SF 6847 (3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile).

Titration of State 4 rat-liver mitochondria at pH 7.2 with the uncoupler 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847) at various concentrations of mitochondria and using various substrates indicates that under optimal conditions less than 0.2 molecule of 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile per respiratory chain is sufficient to induce complete uncoupling. This result suggests that there is not a stoichiometric relationship between uncoupler molecules and cytochrome c oxidase, involved in oxidative phosphorylation, or between the former and phosphorylation assemblies. Experiments on the release by 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile of azide-inhibited respiration of State 3 mitochondria and titrations with 5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide (S13) of State 4 mitochondria at various mitochondrial concentrations confirm this conclusion.

The reduction of artificial electron acceptors at sub-zero temperatures by chloroplasts suspended in fluid media.

1. Chloroplasts can be suspended in aqueous/organic mixtures which are liquid at sub-zero temperatures with a good retention of the ability to reduce artificial electron acceptors. The reduction of ferricyanide and 2,6-dichlorophenolindophenol at temperatures above 0 degrees C is about 50% inhibited by 50% (v/v) ethylene glycol. Higher concentrations cause more extensive inhibition. 2. Different solvents were compared on the basis of their ability to cause a given depression of the freezing point of an aqueous solution. Ethylene glycol caused less inhibition of electron transport than glycerol, which in turn was found to be superior to methanol. 3. The reduction of oxidised 2,3,5,6-tetramethyl-p-phenylenediamine could be measured at -25 degrees C in 40% (v/v) ethylene glycol. Using an acceptor with a high extinction coefficient, methyl purple (a derivative of 2,6-dichlorophenolindophenol) it was possible to observe electron flow at temperatures as low as -40 degrees C in 50% (v/v) ethylene glycol. 4. From studies of the effects of the inhibitors 3(3,4-dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone it is suggested that electron flow from the donor side of Photosystem II to the acceptor side of Photosystem I can occur at temperatures at least as low as -25 degrees C. The ultimate electron donor is presumably water but it was not possible to demonstrate this directly.

Mechanism of proton translocation associated to oxidation of N,N,N',N'-tetramethyl-p-phenylenediamine in rat liver mitochondria.

A kinetic analysis is presented of proton translocation, TMPD+ formation and oxidation of endogenous respiratory carriers during oxygen pulses of TMPD supplemented rat-liver mitochondria. The results show that antimycin-insensitive proton ejection observed under coupled conditions derives from oxidation of endogenous respiratory carriers and re-reduction of TMPD+ by hydrogenated donors and not from proton pumping by cytochrome oxidase as claimed by other investigators. The observations presented provide an example of certain interpretative difficulties in the use of redox mediators and of the methodological approaches that can be used to avoid these.

Activity of proteoliposomes containing cytochrome oxidase in the submitochondrial orientation.

Cytochrome-c oxidase proteoliposomes containing internally trapped cytochrome c can turn over on internal or external cytochrome c. At low TMPD levels the internal activity is significantly lower than the external activity as the functional internal cytochrome c is not fully reduced in the steady state. Increasing TMPD concentration increases the internal rate to equal that of the external enzyme. Internal activity results in the accumulation of TMPD+. Valinomycin increases this accumulation and subsequently FCCP decreases it. In the presence of excess external cytochrome c, the effects of these ionophores are reversed. The internally-facing enzyme is thus capable of generating a delta mu H+ in proteoliposomes as well as in submitochondrial particles.

The oxygen reactive species of cytochrome-c-oxidase: an alternative view.

In a recent review article Babcok and Wikström (Nature, 1992, 356, 301-309) proposed that the species of cytochrome-c-oxidase which binds molecular oxygen during turnover is the so-called mixed valence enzyme, in which the binuclear center cytochrome a3-CuB is reduced, while the cytochrome a/CuA sites are oxidized. This proposal is based on earlier work (Morgan and Wikström, Biochemistry 1991, 30, 948-958) in which it was found that the steady-state reduction levels of cytochrome c and cytochrome a in respiring rat liver mitochondria (sustained by ascorbate and TMPD) are quite different, the latter being much more oxidized than the former; evaluation of the steady-state reduction levels demanded a large correction due to the optical contribution of oxidized TMPD+ which overlaps with the cytochromes. We report below that application of transient spectroscopy and SVD analysis to respiring rat heart myocytes, under conditions in which the contribution of TMPD+ is very small or absent, allows to show that the steady-state reduction levels of cytochrome c and cytochrome a are comparable at all times accessible to measurement in the rapid-scanning stopped-flow spectrophotometer. Our conclusion, in agreement with previous results, is that mixed valence cytochrome-c-oxidase as defined above is not the prevailing oxygen binding species of cytochrome-c-oxidase, unless electron donation to cytochrome c becomes rate limiting.

Interaction of carbonyl cyanide m-chlorophenylhydrazone with the photosystem II acceptor side.

We show that CCCP, known as an uncoupler of photophosphorylation and an ADRY agent, inhibits FeCy photoreduction and coupled O2 evolution by isolated chloroplasts equally (I50 approximately 2 microM), but is practically without effect on the O2 evolution coupled with SiMo reduction within the 0.2-10 microM concentration range. CCCP has no effect on the nanosecond chlorophyll fluorescence in chloroplasts incubated at low light intensity, but decreases it at high light intensity. The electron transfer from reduced TMPD or duroquinol to methylviologen is resistant to CCCP. The efficiency of the CCCP inhibitory action on the FeCy photoreduction depends on the rate of electron flow, which is controlled by the light intensity. The data obtained show that CCCP is oxidized by the photosystem II donor side and is reduced by QP, competing for electrons with FeCy and the cytochrome blf complex.

In vivo control of respiration by cytochrome c oxidase in human cells.

The metabolic control of oxidative phosphorylation (OXPHOS) has attracted increasing attention in recent years, especially due to its importance for understanding the role of mitochondrial DNA mutations in human diseases and aging. Experiments on isolated mitochondria have indicated that a relatively small fraction of each of several components of the electron transport chain is sufficient to sustain a normal respiration rate. These experiments, however, may have not reflected the in vivo situation, due to the possible loss of essential metabolites during organelle isolation and the disruption of the normal interactions of mitochondria with the cytoskeleton, which may be important for the channeling of respiratory substrate to the organelles. To obtain direct evidence on this question, in particular, as concerns the in vivo control of respiration by cytochrome c oxidase (COX), we have developed an approach for measuring COX activity in intact cells, by means of cyanide titration, either as an isolated step or as a respiratory chain-integrated step. The method has been applied to a variety of human cell types, including wild-type and mtDNA mutation-carrying cells, several tumor-derived semidifferentiated cell lines, as well as specialized cells removed from the organism. The results obtained strongly support the following conclusions: (i) the in vivo control of respiration by COX is much tighter than has been generally assumed on the basis of experiments carried out on isolated mitochondria; (ii) COX thresholds depend on the respiratory fluxes under which they are measured; and (iii) measurements of relative enzyme capacities are needed for understanding the role of mitochondrial respiratory complexes in human physiopathology.

Respiratory electron transfer activity in an asolectin-isooctane reverse micellar system.

Bovine heart submitochondrial particles (SMP) were solubilized in an asolectin isooctane reverse micellar system and the functionality of the respiratory chain was tested by spectroscopic and amperometric techniques. Electron transfer rate supported by NADH was very slow as evidenced by the low cytochrome reduction levels attained over long incubation periods. In the presence of KCN, NADH caused 34% and 12.5% reduction of the cytochromes aa3 and c, respectively, and negligible reduction of cytochrome b. Supplementation of the system with menadione rose the NADH-dependent reduction of all the cytochromes to levels that were close to the total content. However, no measurable O2 uptake activity took place in the presence of NADH plus menadione, or with ascorbate (or NADH) plus TMPD reducing systems. Therefore, it is suggested that in the organic medium, electron transfer from NADH to O2 is arrested at the terminal oxidase step. Cytochrome oxidase reduced by ascorbate (or NADH) plus TMPD seems to be trapped in its half reduced state (ie, a2+ a3(3+)). Although it is poorly reactive with O2, it can transfer electrons back to cytochrome c and TMPD. The electron transfer block to O2 was overcome when PMS was used instead of TMPD. This seems to be due to the recognized capacity of PMSH2 to carry out simultaneous reduction of both a CuA and a3 CuB redox centers of cytochrome oxidase. The cytochrome oxidase reaction in the organic solvent was highly sensitive to KCN (Ki 1.9 microM) and showed bell-shaped kinetics towards the PMS concentration and a sigmoidal response to water concentration, reaching its maximal turnover number (18 s-1) at 4 mM PMS and 1.1% (v/v) water.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of isoquinoline derivatives structurally related to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on mitochondrial respiration.

Isoquinoline derivatives exert 1-methyl-4-phenylpyridinium (MPP+)-like activity as inhibitors of complex I and alpha-ketoglutarate dehydrogenase activity in rat brain mitochondrial fragments. We now examine the ability of 19 isoquinoline derivatives and MPP+ to accumulate and inhibit respiration in intact rat liver mitochondria, assessed using polarographic techniques. None of the compounds examined inhibited respiration supported by either succinate + rotenone or tetramethylparaphenylenediamine (TMPD) + ascorbate. However, with glutamate + malate as substrates, 15 isoquinoline derivatives and MPP+ inhibited state 3 and, to a lesser extent, state 4 respiration in a time-dependent manner. None of the isoquinoline derivatives were more potent than MPP+. 6,7-Dimethoxy-1-styryl-3,4-dihydroisoquinoline uncoupled mitochondrial respiration. Qualitative structure-activity relationship studies revealed that isoquinolinium cations were more active than isoquinolines in inhibiting mitochondrial respiration; these, in turn, were more active than dihydroisoquinolines and 1,2,3,4-tetrahydroisoquinolines. Three-dimensional quantitative structure-activity relationship studies using Comparative Molecular Field Analysis showed that the inhibitory potency of isoquinoline derivatives was determined by steric, rather than electrostatic, properties of the compounds. A hypothetical binding site was identified that may be related to a rate-limiting transport process, rather than to enzyme inhibition. In conclusion, isoquinoline derivatives are less potent in inhibiting respiration in intact mitochondria than impairing complex I activity in mitochondrial fragments. This suggests that isoquinoline derivatives are not accumulated by mitochondria as avidly as MPP+. The activity of charged and neutral isoquinoline derivatives implicates both active and passive processes by which these compounds enter mitochondria, although the quaternary nitrogen moiety of the isoquinolinium cations favours mitochondrial accumulation and inhibition of respiration. These findings suggest that isoquinoline derivatives may exert mitochondrial toxicity in vivo similar to that of MPTP/MPP+.

Characterization of electron donation to cytochrome c-555 in Chromatium vinosum from ferrocyanide, tetramethylphenylenediamine and reduced dimethylquinone. Effects of redox potential, pH and salt concentration.

1. The dependences of the reduction of ferricytochrome c-555 in the reaction center-cytochrome c complex on the redox potential and pH were investigated using N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), ferrocyanide, and reduced 2,5-dimethyl-p-quinone as electron donors. 2. In the reduction of cytochrome c-555 by TMPD, the unprotonated form was the exclusive electron donor to the cytochrome with a second-order rate constant of 1.0 X 10(5) M-1.s-1. 3. Ferrocyanide reduced cytochrome c-555 slowly with a rate constant of 7.8 X 10(3) M-1.s-1 at infinite salt concentration. The value of -5.2 X 10(-4) elementary charge/A2 was estimated as the surface charge density in the vicinity of cytochrome c-555 by analyzing the salt effect on the cytochrome reduction using the Gouy-Chapman theory. 4. The characteristics of the dependences of the reduction of cytochrome c-555 by reduced 2,5-dimethyl-p-quinone on the redox potential and pH were well explained by the redox potential and pH dependences of the formation of the semiquinone. In the neutral-to-alkaline pH range the anionic semiquinone was the main electron-donating species with a second-order rate constant of 6.0 X 10(7) m-1.s-1.

A novel terminal oxidase, cytochrome baa3 purified from aerobically grown Pseudomonas aeruginosa: it shows a clear difference between resting state and pulsed state.

A novel type of cytochrome c oxidase was purified to homogeneity from Pseudomonas aeruginosa which was grown aerobically. The purified oxidase contained two molecules of heme a, two atoms of copper, and one molecule of protoheme per molecule. One of the two heme a molecules in the oxidase reacted with carbon monoxide, so that the enzyme was of baa3-type. The oxidase molecule was composed of three subunits with molecular weights of 38,000, 57,000, and 82,000. Although the oxidase oxidized ferrocytochrome c-550 obtained from the bacterial cells grown aerobically, the oxidizing activity was not high. The "resting form" and the "pulsed form" of the oxidase were observed clearly with this enzyme, and the transition from the resting form to the pulsed form was accompanied by a distinct change of the enzymatic activity. The difference in the kinetics of the catalytic reactions between the two forms is discussed.