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.

Comparative antioxidant activities and synergism of resveratrol and oxyresveratrol.

Resveratrol (1) and oxyresveratrol (2) are phytoalexins with antioxidant activities (AAs) and proposed effects against several pathological processes. The main objective of this study was to provide a novel, comparative assessment of their AAs, and to test for potential synergism in their combined activities, or in combination with another phytochemical antioxidant, curcumin (3). The phytochemicals were tested at 10 µM total concentrations in a heme-based assay that involved, as the final step, quantification of tetramethyl-phenylene-diamine oxidation. Significant AAs were observed for both 1 and 2, 27-33% inhibition of oxidation (p < 0.05 relative to non-phytochemical control). The combination of 1 and 2 in the same assay (5 µM each) suggested a moderate synergistic effect of about 10% (41% inhibition of oxidation by 1/2 under the same conditions as for 1 and 2 separately). Combinations of 1/3 and 2/3 were also synergistic, but 1/3 had a two-fold greater AA (p < 0.05) than 2/3 (or 1/2). Our results indicate that (i) 1 and 2 are effective antioxidants in the assay, (ii) in combination, their AAs can synergise, and (iii) in relation to 2, 1 has a much greater synergistic potential with 3. The latter suggests different synergy mechanisms of the curcuminoid with each of the two stilbene phytoalexins.

Using N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) to assay cyclooxygenase activity in vitro.

Prostaglandin endoperoxide synthase (PGH synthase), also known as cyclooxygenase (COX), was identified over 30 years ago and is the key enzyme in the pathway by which arachidonic acid is converted to the range of biologically active lipid mediators known as the prostanoids that participate in numerous physiological processes. The need for the development of new and improved COX inhibitors as potential therapeutics also drives the need for rapid, reliable, and inexpensive assays of COX activity. Colorimetric assays are often the preferred methods of enzyme analysis since they may be readily adapted to simple microplate formats that require relatively inexpensive and widely available instrumentation. The use of N,N,N cent,N cent-tetramethyl-p-phenylenediamine (TMPD) in high throughput microplate assays of COX activity could become the approach of choice in the screening of potential therapeutics that inhibit COX activity in vivo. Considering that TMPD is also a potential substrate for most, if not all, heme peroxidases, it is anticipated that this agent could find increasing application in the future.

Evidence for the presence of prostaglandin H synthase like enzyme in female Setaria cervi and its inhibition by diethylcarbamazine.

Experimental evidence has shown that Setaria cervi a bovine filarial parasite contains significant amount of prostaglandin H synthase like activity in the somatic extract of its different life stages. A protein with characteristics of prostaglandin H synthase was purified to homogeneity from female somatic extract using a combination of affinity and gel filtration chromatography. Molecular weight of purified enzyme was 70kDa as determined by SDS-PAGE. Purified enzyme showed high activity with arachidonic acid and TMPD substrates suggests the presence of both cyclooxygenase and peroxidase activity in enzyme. Fluorescence spectroscopy and hemin-associated peroxidase activity confirmed presence of heme in purified enzyme. The K(m) and V(max) values using arachidonic acid were determined to be 79+/-1.5microM and 0.165+/-0.2U/ml, respectively. Further, indomethacin and aspirin, specific inhibitors for PGHS, significantly inhibited the enzyme activity. Diethylcarbamazine, an antifilarial drug inhibited the microfilarial PGHS like activity as well as their motility. Here we are reporting for the first time PGHS like activity in filarial parasite and its inhibition with DEC which provide that this enzyme could be used as a drug target.

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.

Arginine 391 in subunit I of the cytochrome bd quinol oxidase from Escherichia coli stabilizes the reduced form of the hemes and is essential for quinol oxidase activity.

The cytochrome bd quinol oxidase is one of two respiratory oxidases in Escherichia coli. It oxidizes dihydroubiquinol or dihydromenaquinol while reducing dioxygen to water. The bd-type oxidases have only been found in prokaryotes and have been implicated in the survival of some bacteria, including pathogens, under conditions of low aeration. With a high affinity for dioxygen, cytochrome bd not only couples respiration to the generation of a proton motive force but also scavenges O(2). In the current work, the role of a highly conserved arginine residue is explored by site-directed mutagenesis. Four mutations were made: R391A, R391K, R391M, and R391Q. All of the mutations except R391K result in enzyme lacking ubiquinol oxidase activity. Oxidase activity using the artificial reductant N,N,N',N'-tetramethyl-p-phenylenediamine in place of ubiquinol was, however, unimpaired by the mutations, indicating that the catalytic center where O(2) is reduced is intact. UV-visible spectra of each of the mutant oxidases show no perturbations to any of the three heme components (heme b(558), heme b(595), and heme d). However, spectroelectrochemical titrations of the R391A mutant reveal that the midpoint potentials of all of the heme components are substantially lower compared with the wild type enzyme. Since Arg(391) is close to Met(393), one of the axial ligands to heme b(558), it is to be expected that the R391A mutation might destabilize the reduced form of heme b(558). The fact that the midpoint potentials of heme d and heme b(595) are also significantly lowered in the R391A mutant is consistent with these hemes being physically close together on the periplasmic side of the membrane.

Mitochondrial oxidative phosphorylation is defective in the long-lived mutant clk-1.

The long-lived mutant of Caenorhabditis elegans, clk-1, is unable to synthesize ubiquinone, CoQ(9). Instead, the mutant accumulates demethoxyubiquinone(9) and small amounts of rhodoquinone(9) as well as dietary CoQ(8). We found a profound defect in oxidative phosphorylation, a test of integrated mitochondrial function, in clk-1 mitochondria fueled by NADH-linked electron donors, i.e. complex I-dependent substrates. Electron transfer from complex I to complex III, which requires quinones, is severely depressed, whereas the individual complexes are fully active. In contrast, oxidative phosphorylation initiated through complex II, which also requires quinones, is completely normal. Here we show that complexes I and II differ in their ability to use the quinone pool in clk-1. This is the first direct demonstration of a differential interaction of complex I and complex II with the endogenous quinone pool. This study uses the combined power of molecular genetics and biochemistry to highlight the role of quinones in mitochondrial function and aging.

[The cytochrome cbo from the obligate methylotroph Methylobacillus flagellatus KT is a cytochrome-c oxidase]. / Tsitochrom cbo obligatnogo metilotrofa Methylobacillus flagellatus KT iavliaetsia tsitokhrom-c-oksidazoi.

The oxidase cho of Methylobacillus flagellatus KT was purified to homogeneity by nondenaturing gel electrophoresis, and the kinetic properties and substrate specificity of the enzyme were studied. Ascorbate and ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) were oxidized by cbo with a pH optimum of 8.3. When TMPD served as electron donor for the oxidase cho, the optimal pH (7.0 to 7.6) was determined from the difference between respiration rates in the presence of ascorbate/TMPD and of only ascorbate. The kinetic constants, determined at pH 7.0, were as follows: oxidation by the enzyme of reduced TMPD at pH 7.0 was characterized by KM = 0.86 mM and Vmax = 1.1 mumol O2/(min mg protein), and oxidation of reduced cytochrome c from horse heart was characterized by KM = 0.09 mM and Vmax = 0.9 mumol O2/(min mg protein) Cyanide inhibited ascorbate/TMPD oxidase activity (Ki = 4.5-5.0 microM). The soluble cytochrome cH (12 kDa) partially purified from M. flagellatus KT was found to serve as the natural electron donor for the oxidase cbo.

A cytochrome c-enhanced peroxidation reaction with potential use in screening dietary antioxidants.

Reactive oxygen species (ROS) that result from events such as cellular respiration can cause damage to biological molecules and tissues. A variety of endogenous and dietary antioxidants function in moderating the extent of oxidative damage in the body. In this report, a pro-oxidant system is presented as an assay for screening possible antioxidant activities of dietary factors. The assay reaction involves peroxidatic oxidation of the redox indicator N,N,N',N'-tetramethyl-1,4-phenylenediamine (TMPD). It is shown that the reaction rate is enhanced by up to 10-fold in the presence of cytochrome c (cyt c), a mitochondrial electron transport protein. The extent to which selected dietary antioxidant factors inhibit the cytochrome c-enhanced peroxidatic oxidation of TMPD is also reported. Considering the known pathological consequences of mitochondrial membrane disruption and cytochrome c release in the cell, this reaction and assay may be of pathological and therapeutic relevance.

Heat shock protein 72 binds and protects dihydrofolate reductase against oxidative injury.

Although heat shock protein Hsp72 confers resistance to oxidative injury, the mechanisms are unknown. These studies demonstrate that Hsp72 protects dihydrofolate reductase (DHFR) against injury caused by the thiol oxidant monochloramine (NH(2)Cl). When exposed to NH(2)Cl, DHFR catalytic activity is impaired and SDS-PAGE migration retarded. These may be blocked by prior addition of Hsp72 or the folate analog methotrexate. Methotrexate binding to DHFR is diminished by oxidant treatment, preventable by prior Hsp72 incubation. Hsp72 also protects DHFR in IEC-18 cells following oxidant exposure. Hsp72 co-immunoprecipitates with DHFR, especially after partial oxidation. The DHFR-Hsp72 interaction is modulated by cofactor/substrate binding for both Hsp72 (ATP) and DHFR (methotrexate). Thiol oxidation of DHFR increases susceptibility for tryptic proteolysis. Preincubation of DHFR with Hsp72 prevents the NH(2)Cl-induced sensitivity to proteolysis. Thus, Hsp72 binds DHFR through enhanced protein-chaperone interactions upon oxidant exposure, a process that may protect against irreversible modification of DHFR catalytic and structural integrity.

Aeropyrum pernix K1, a strictly aerobic and hyperthermophilic archaeon, has two terminal oxidases, cytochrome ba3 and cytochrome aa3.

Aeropyrum pernix K1 is a strictly aerobic and hyperthermophilic archaeon that thrives even at 100 degrees C. The archaeon is quite interesting with respect to the evolution of aerobic electron transport systems and the thermal stability of the respiratory components. An isolated membrane fraction was found to oxidize bovine cytochrome c. The activity was solubilized in the presence of detergents and separated into two fractions by successive chromatography. Two cytochrome oxidases, designated as CO-1 and CO-2, were further purified. CO-1 was a ba(3)-type cytochrome containing at least two subunits. Chemically digested fragments of CO-1 revealed a peptide with a sequence identical to a part of a putative cytochrome oxidase subunit I encoded by the gene ape1623. CO-2, an aa(3)-type cytochrome, was present in lower amounts than CO-1 and was immunologically identified as a product of aoxABC gene (DDBJ accession no. AB020482). Both cytochromes reacted with carbon monoxide. The apparent K(m) values of CO-1 and CO-2 for oxygen were 5.5 and 32 micro M, respectively, at 25 degrees C. The terminal oxidases CO-1 and CO-2 phylogenetically correspond to the SoxB and SoxM branches, respectively, of the heme-copper oxidase tree.

The aerobic electron transport system of Eikenella corrodens.

The respiratory system of the fastidious beta-proteobacterium Eikenella corrodens grown with limited oxygen was studied. Membranes showed the highest oxidase activity with ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) or succinate and the lowest activity with NADH and formate. The presence of a bc1-type complex was suggested by the inhibition exerted by 2-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), myxothiazol, and antimycin A on respiration with succinate and by the effect of the latter two inhibitors on the succinate-reduced difference spectra. Respiration with succinate or ascorbate-TMPD was abolished by low KCN concentrations, suggesting the presence of a KCN-sensitive terminal oxidase. Cytochromes b and c were spectroscopically detected after reduction with physiological or artificial electron donors, whereas type a and d cytochromes were not detected. The CO difference spectrum of membranes reduced by dithionite and its photodissociation spectrum (77 K) suggested the presence of a single CO compound that had the spectral features of a cytochrome o-like pigment. High-pressure liquid chromatography analysis of membrane haems confirmed the presence of haem B; in contrast, haems A and O were not detected. Peroxidase staining of membrane type c cytochromes using SDS-PAGE revealed the presence of five bands with apparent molecular masses of 44, 33, 30, 26, and 14 kDa. Based on our results, a tentative scheme of the respiratory chain in E. corrodens, comprising (i) dehydrogenases for succinate, NADH, and formate, (ii) a ubiquinone, (iii) a cytochrome bc1, and (iv) a type-cbb' cytochrome c oxidase, is proposed.

Mitochondrial respiration at low levels of oxygen and cytochrome c.

In the intracellular microenvironment of active muscle tissue, high rates of respiration are maintained at near-limiting oxygen concentrations. The respiration of isolated heart mitochondria is a hyperbolic function of oxygen concentration and half-maximal rates were obtained at 0.4 and 0.7 microM O(2) with substrates for the respiratory chain (succinate) and cytochrome c oxidase [N,N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD)+ascorbate] respectively at 30 degrees C and with maximum ADP stimulation (State 3). The respiratory response of cytochrome c-depleted mitoplasts to external cytochrome c was biphasic with TMPD, but showed a monophasic hyperbolic function with succinate. Half-maximal stimulation of respiration was obtained at 0.4 microM cytochrome c, which was nearly identical to the high-affinity K(')(m) for cytochrome c of cytochrome c oxidase supplied with TMPD. The capacity of cytochrome c oxidase in the presence of TMPD was 2-fold higher than the capacity of the respiratory chain with succinate, measured at environmental normoxic levels. This apparent excess capacity, however, is significantly decreased under physiological intracellular oxygen conditions and declines steeply under hypoxic conditions. Similarly, the excess capacity of cytochrome c oxidase declines with progressive cytochrome c depletion. The flux control coefficient of cytochrome c oxidase, therefore, increases as a function of substrate limitation of oxygen and cytochrome c, which suggests a direct functional role for the apparent excess capacity of cytochrome c oxidase in hypoxia and under conditions of intracellular accumulation of cytochrome c after its release from mitochondria.

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.

Metabolic fate of chemical mixtures. I. "Shuttle Oxidant" effect of lipoxygenase-generated radical of chlorpromazine and related phenothiazines on the oxidation of benzidine and other xenobiotics.

Many carcinogens, mutagens, teratogens, and other toxicants are known to be oxidized by lipoxygenases to potentially deleterious free radical intermediates. In this study, we tested for the first time the possibility that certain efficient substrates for lipoxygenase produce shuttle oxidants that stimulate the generation of reactive species from other chemicals. To evaluate the hypothesis, we investigated the metabolic interaction of two well-known substrates, chlorpromazine and benzidine, which have been shown to be oxidized by soybean lipoxygenase in the presence of hydrogen peroxide. The evidence presented here clearly indicates that the chlorpromazine cation radical generated by the lipoxygenase triggers a rapid oxidation of benzidine to benzidine diimine. Under the experimental conditions employed, the metabolic interaction resulted in a 42-fold stimulation in the rate of benzidine oxidation. The magnitude of stimulation of benzidine oxidation exhibited a dependence on the pH of the reaction medium, amount of the enzyme, and concentration of chlorpromazine, benzidine, and hydrogen peroxide. A number of other phenothiazines were also found to stimulate benzidine oxidation, albeit to a lesser degree. The chlorpromazine cation radical stimulated the oxidation of all six other xenobiotics tested. The highest stimulation (94-fold) was noted with tetramethyl phenylenediamine oxidation to the Wursters blue radical, while the lowest stimulatory response (2-fold) was observed with guaiacol. Preliminary data suggest that purified human term placental lipoxygenase also displays a similar stimulatory response in the benzidine oxidation in the presence of chlorpromazine. Although the toxicological significance of these in vitro findings remains to be established, it is worth pondering whether such a synergistic interaction occurs in humans in vivo. Teratogenesis Carcinog. Mutagen. 20:195-208, 2000.

Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I.

We report here a new mitochondrial regulation occurring only in intact cells. We have investigated the effects of dimethylbiguanide on isolated rat hepatocytes, permeabilized hepatocytes, and isolated liver mitochondria. Addition of dimethylbiguanide decreased oxygen consumption and mitochondrial membrane potential only in intact cells but not in permeabilized hepatocytes or isolated mitochondria. Permeabilized hepatocytes after dimethylbiguanide exposure and mitochondria isolated from dimethylbiguanide pretreated livers or animals were characterized by a significant inhibition of oxygen consumption with complex I substrates (glutamate and malate) but not with complex II (succinate) or complex IV (N,N,N',N'-tetramethyl-1, 4-phenylenediamine dihydrochloride (TMPD)/ascorbate) substrates. Studies using functionally isolated complex I obtained from mitochondria isolated from dimethylbiguanide-pretreated livers or rats further confirmed that dimethylbiguanide action was located on the respiratory chain complex I. The dimethylbiguanide effect was temperature-dependent, oxygen consumption decreasing by 50, 20, and 0% at 37, 25, and 15 degrees C, respectively. This effect was not affected by insulin-signaling pathway inhibitors, nitric oxide precursor or inhibitors, oxygen radical scavengers, ceramide synthesis inhibitors, or chelation of intra- or extracellular Ca(2+). Because it is established that dimethylbiguanide is not metabolized, these results suggest the existence of a new cell-signaling pathway targeted to the respiratory chain complex I with a persistent effect after cessation of the signaling process.

Kinetic analysis of substrate inhibition in nitric oxide reductase of Paracoccus denitrificans.

The current kinetic model for the nitric oxide reductase reaction (Girsch, P., and de Vries, S. (1997) Biochim. Biophys. Acta 1318, 202-216) does not involve the concentration of an electron donor. Here we introduce this variable and show, both theoretically and experimentally, its role in determining the extent of substrate inhibition by the excess of nitric oxide. NO is found to inhibit competitively with the electron donor, possibly by binding to the oxidized form of the enzyme. The observed partial character of the inhibition is tentatively explained by a slow reduction of the non-productive NO complex.