Oxidation titrations, anaerobic

The oxidation-reduction potential (E6) of adrenodoxin is 164 mV at pH 7.4 and 26° C by the potentiometric titration method with dithionite in a nitrogen atmosphere (29). However, Estabrook and his colleagues obtain a value of —196 mV in the determination of the potential anaerobically by titration with NADPH plus adrenodoxin reductase using dyes with electromotive activity. The difference in value between the two... 

The reduction state of the pterin was a point of uncertainty throughout these studies of molybopterin derivatives. The absence of fluorescence in anaerobic molybdopterin samples suggested a reduced pterin. Redox titration of XO and SO both indicated that the pterin could undergo a two-electron oxidation reaction (73, 74). Sulfite oxidase, for example, produced the fluorescence characteristic of an oxidized pterin after addition of 2 equiv of ferricyanide. However, titrating XO was problematic due to interfering redox processes of the iron-sulfur clusters. 

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. 

FiO. 11. Ckwnplex formation between the 2-electron-reduced (EHi) form of E. coli glutathione reductase and NADPH. EH2 was produced by anaerobic reduction with borohydride time was allowed for the slight excess of borohydride to react with water before beginning the titration with NADPH. 1, Oxidized 2, EHi 3, EHa-f-0.45 equivalent NADPH 4, EHi-1-0.90 equivalent NADPH and 5, EH -1-2.75 equivalents NADPH. 

There now seems little doubt that complete oxidation or reduction of the isolated oxidase under anaerobic conditions requires four electron equivalents (I64, 165). When the potentiometric titration is followed spectrophotometrically (at 605-630 nm), high potential (at 350-375 mV) and low potential (210-230 mV) hemes (Table VIII) are indicated... 

Cells, membrane preparations, or isolated RC complexes were used for the determination of cytochromes oxidized by flash excitation under aerobic conditions and anaerobic conditions in the presence of ascorbate and DAD. In some species, redox titrations of cytochromes (flash-induced changes and total contents) were also performed. 

It was possible that the decline in Pgyo oxidation was brought about by an alteration in the redox potential of a reaction centre. Redox titrations of the rate of Pg70 oxidation showed that neither aerobic or anaerobic treatment significantly affected the optimum operating potential of the system (Fig 3). A number of molecules such as DBMIB are known to quench chi fluorescence by a... 

Fig 3. An oxidative redox titration of the rate of PgjQ oxidation before ( — > preillumination, after 15 min aerobic treatment (O—O) and after 4 hr anaerobic treatment (A—A),... 

Fig.10 Potentiometric titration of MCAD (10.7 (iM) in the presence of HD-CoA (181 tM). MV++ (100 tM) serves as a redox mediator. Redox indicators are pyocyanine (5.0 pM) and indigo disulfonate (2.5 pM). Titration was performed under anaerobic conditions at 25 °C in 50 mM potassium phosphate buffer, pH 7.6. Intermediate spectra have been removed for clarity. The spectrum of the oxidized MCAD HD-CoA complex is shown by curve 1. Curves 2-6 show the complex at = —30, —44, —51, —62, and —79 mV versus SHE. Curve 7 is the spectrum of the fully reduced MCAD bound to HD-CoA. Inset Nernst plot indicating an E° = —0.052 V and n = 1.6.

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