Electron phenazine methosulfate

The reduction of tetrazolium salts by NADH is greatly accelerated by electron transfer agents (ETAs) such as phenazine methosulfate (PMS 233) or its derivatives.451-454 Other classes of ETAs such as quinones.455,456 ferricinium,457 phenothiazine,458 the viologens,459 acridiniums,460 and phe-nazinium or quinoxalinium salts461 as well as the enzyme diaphorase462 have been used. 

Recently a colorimetric method for estimation of erythrocytic G-6-PDH was described (El). This procedure is based upon the interaction of phenazine methosulfate as electron carrier between NADPH2 formed in the reaction and dichloroindophenol, the rate of the reduction of the latter compound being followed at 620 mp. 

Abbreviations ATP, adenosine-5 -triphosphate EPR, electron paramagnetic resL nance EXAFS, extended X-ray absorption 6ne structure Hb, hemoglobin Hb, oxidized (met-) hemoglobin NADH, reduced form of nicotinamide-adenosine dinucleotide PMS, phenazine methosulfate (methylsulfate salt of N-methylphenazonium cation) TMPD, N.N.N N -tetramethylphenylene-1,4 diaminium dication SDS-PAOE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 

The activity of complex II (succinate dehydrogenase) is measured as the succinate-dependent reduction of decylubiquinone, which is in turn recorded spectro-photometrically through the reduction of dichlorophenol indophenol at 600 nm (e 19,100-M -cm Fig. 3.8.5). In order to ensure a linear rate for the activity, the medium is added with rotenone, ATP, and a high concentration of succinate. As noticed previously for complex I, decylubiquinone is not a perfect acceptor for electrons from the membrane-inserted complex II [70]. Malonate, a competitive inhibitor of the enzyme, is used to inhibit it. Rather than decylubiquinone, phenazine methosulfate can be utilized, which diverts the electrons from the complex before they are conveyed through subunits C and D, therefore allowing measurement of the activity of subunits A and B. 

Several findings in the above results are not consistent with earlier reports (Yoshikawa et al., 1995 Van Gelder, 1966 Tiesjema et al., 1973 Schroedl and Hartzell, 1977 Babcock et al., 1978 Blair et al., 1986 Steffens et al., 1993). It has been widely accepted that four electron equivalents are sufficient for complete reduction of the fuUy oxidized enzyme as prepared. However, most of the previous titrations were performed in the presence of electron transfer mediators. In the presence of electron transfer mediators, such as phenazine methosulfate (PMS) under anaerobic conditions, the bovine heart enzyme purified with crystallization also showed a four-electron reduction without the initial lag phase as observed in Fig. 9. A catalytic amount of PMS induced a small spectral change corresponding to the initial lag phase. These results suggest that electron transfer mediators in other titration experiments also induce autoreductions to provide the enzyme form that receives four electrons for the complete reduction. 

NMR, nuclear magnetic resonance mV, millivolt pCMB, p hloromercuribenzoate pCMS, p-chloromercuriphenyl sulfonate PMS, phenazine methosulfate EDTA, ethylenediaminetetraacetate SDS, sodium dodecyl sulfate TTFA, 2-thenoyltriflu-oroacetone ETP, submitochondrial (electron transfer) particles MVH, reduced methyl viologen DEAE-cellulose, diethylaminoethyl cellulose succ, succinate APS, adenosine S -phosphosulfate PAPS, 3 -phosphoadenosine 5 -phosphosulfate. Other abbreviations are standard [see JBC 244, 2 (1969)]. 

Physical measurements support a molecular weight of approximately 200,000. This value is also in accord with gel exclusion studies on Sephadex G-200. Thus the enzyme contains 1 mole of flavin and 4 g-atoms of nonheme iron per mole.. . . The sedimentation velocity of the beef heart enzyme at 10-15 mg protein/ml is 6.5 S. . . This preparation could oxidize succinate in the presence of ferricyanide or phenazine methosulfate (PMS) as electron acceptor but was unable to transfer elec-to be unable to interact with the respiratory chain. 

D-Lactate cytochrome c reductase can oxidize D-2-hydroxymonocar-boxylic acids, but only D-lactate and D-2-hydroxybutyrate are oxidized at appreciable rates. The enzyme exhibits a similar high specificity for electron acceptors. It reacts with cytochrome c and phenazine methosul-fate as electron acceptors, but not with ferricyanide, methylene blue, 2,6-dichloroindophenol, and menadione 308, 312, 313). With D-lactate as substrate and at Fmax with respect to acceptor, phenazine methosulfate is reduced at 30° eight times as fast as cytochrome c 308). The values at 30° and pH 7.5 are D-lactate, 0.29 mM n-2-hydroxybutyrate,... 

The rates of cyclic photophosphorylation around PS I catalysed by the natural catalysts are rather low, about one order of magnitude lower than those of linear electron transport [59], while they are very high when artificial electron carriers, such as phenazine methosulfate, are added to the system. Cyclic photophosphorylation has been shown to occur in intact leaves [65] and algae [66]. 

Assay of lactate dehydrogenase illustrates this technique. This enzyme catalyzes the oxidation of lactate, yielding pyruvate and NADH as shown in Figure 6-17. NADH produced in this reaction can be used along with the intermediate electron carrier, phenazine methosulfate (PMS), to... 

Fig. 12.8. Schematic representation of events occurring during biogenesis of photosystem I reaction center. The subunits are designated as I to VII, the abbreviations are Ferr, ferredoxin P.C., plas-tocyanin A, Aj, Aj and A4, primary, secondary, tertiary and quaternary electron acceptors PMS, phenazine methosulfate DAD, diaminodurine.

Visual localization of electrophoretically separated LDH isoenzymes is accomplished by the reduction of nitro-tetrazolium blue as the electron acceptor (terminal) in a medium containing phenazine methosulfate and NAD. The linked reaction is as follows ... 

Complex III - Complex III contains a diversity of electron carrying proteins. They include cytochrome b, iron sulfur centers, and cytochrome cl. Cytochrome b is the first of the heme-carrying proteins (Figure 15.6) involved in electron transport. Passage of electrons from cytochrome b to the iron sulfur centers can be blocked by antimycin A. Also, the artificial electron acceptor phenazine methosulfate can accept electrons from cytochrome b and 2,6-dichlorophenol-indophenol can accept electrons from the iron sulfur proteins (Figure 15.9). The crystal structure of the redox components of complex III from bovine heart mitochondria is shown in Figure 15.16... 

The movement of electrons through the electron carrying proteins of the inner mitochondrial membrane is shown in Figure 15.9. Also shown are inhibitors of electron movement at their point of action and the sites where artificial electron acceptors can accept electrons from the electron transport system. Specific inhibitors shown in Figure 15.9 are rotenone, amytal, antimycin A, cyanide, azide, and carbon monoxide. The artificial electron acceptors are methylene blue, phenazine methosulfate, 2,6-indophenol, tetramethyl-p-phenylene diamine, and ferricyanide. 

The failure of a generation of investigators to obtain soluble succinic dehydrogenase preparations was caused by the unusually fastidious requirements of this enzyme. Of the ordinary electron acceptors, including methylene blue and dichlorophenolindophenol, none supports the oxidation of succinate. Phenazine methosulfate was found to be reduced by solubilized preparations the enzyme is extracted from acetone powders and was undoubtedly present in many extracts, where it was not detected for lack of a suitable oxidant. A preparation from yeast was reported... 

LIST OF ABBREVIATIONS ACP, acyl carrier protein 5ALA, 5-aminolevulinic acid AU, 6-azauracyl CAP, chloramphenicol CHI, cycloheximide DCIP, 2,6-dichlorophenolindo-phenol DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea DG, digalactosyl-diglyceride DPC, diphenylcarbazide ETS, electron-transport system FDPase, alkaline fructose 1,6-diphosphate phosphatase G-3-P, glyceraldehyde-3-phosphate MG, monogalactosyldiglycer-ide PSI, PSII, photosystems I and II P700, active center of photosystem I PMS, phenazine methosulfate Q, photosystem II quencher Ru-l,5-diP, ribulose-1,5-diphosphate SDS, sodium dodecylsulfate SL, sulfolipid. 

The spectrophotometric methods described earlier were used to determine the optimum pH for the isoenzymes (Schabort et al., 1971). Cytochrome c [together with a small amount of phenazine methosulfate (PMS) as intermediate electron acceptor] was particularly useful for determinations below pH 6.4, because the absorbance of DCIP at 600 nm decreases rapidly below this value. The five isoenzymes showed the same optimum pH of 6.8 for both the dehydrogenation and the total conversion of jSCA into aCA. The temperature stability of the five isoenzymes was essentially the same. They lost all their activity after heat treatment for 10 min at 75.5°C, but retained 70% of their activity after 10 min at 55" C. 

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