Materials and Reagents

1. 0.2 µm filter (Corning) (Sigma-Aldrich, catalog number: CLS431229 )
2. Potassium phosphate dibasic, anhydrous (Sigma-Aldrich, catalog number: 795496 )
3. Potassium phosphate monobasic, anhydrous (Sigma-Aldrich, catalog number: 795488 )
4. Xanthine (Sigma-Aldrich, catalog number: X7375 )
5. Xanthine oxidase, lyophilized powder (Sigma-Aldrich, catalog number: X4376 )
6. E. coli SdhA
   E. coli SdhA was isolated with an N-terminally fused His-tag as described in Maklashina et al., 2018.
7. Glucose (Sigma-Aldrich, catalog number: G8270 )
8. Catalase (Sigma-Aldrich, catalog number: C30 )
9. Glucose oxidase (Sigma-Aldrich, catalog number: G2133 )
10. Potassium indigotetrasulfonate (ITS) (Sigma-Aldrich, catalog number: 340596 )
11. Benzyl viologen (Sigma-Aldrich, catalog number: 271845 ) or Methyl Viologen (Sigma-Aldrich, catalog number: 856177 )
12. Flavoprotein, isolated SdhA (see the expression and purification method; Maklashina et al., 2018)
13. Ultra High Purity 5.0 Grade Argon (Airgas, catalog number: AR UHP300 ) or Ultra High Purity 5.0 Grade Nitrogen (Airgas, catalog number: NI UHP300 )
14. Sodium Dithionite (Sigma-Aldrich, catalog number: 71699 )
15. Apiezon High Vacuum grease type L (Fisher Scientific, catalog number: 50-365-186 )
16. Nitrogen/argon, gas
17. Toluylene blue
18. Phenazine methosulfate
19. Thionine
20. Methylene blue
21. Pyocyanin
22. Indigotetrasulfonate
23. Resorufin
24. Indigotrisulfonate
25. Indigodisulfonate
26. 2-Hydroxy-1,4 naphthoquinone
27. Cresyl violet
28. Anthraquinone-2,6-disulphonate
29. Anthraquinone-2-sulfonate
30. Phenosafranine
31. Safranine T
32. 50 mM potassium phosphate, pH 7.0 (see Recipes)
33. Xanthine solution, 10 mM (see Recipes)
34. Xanthine oxidase solution (see Recipes)
35. Glucose solution, 1 M (see Recipes)
36. Catalase solution (see Recipes)
37. Glucose oxidase solution (see Recipes)
38. Reference dyes solutions (see Recipes)
39. Benzyl viologen or methyl viologen (see Recipes)
    

Equipment

1. Spectrophotometer (for example, Agilent 8453 UV-visible spectrophotometer capable of scanning between 300 and 800 nm, with a spectral resolution of 1 nm)
   Temperature control is supplied by a Peltier unit from Agilent. Any means to maintain a constant temperature is appropriate (i.e., cuvette holder where the temperature is controlled by a water bath, for example).
2. An appropriate cuvette to conduct the reaction under strict anaerobic conditions (Figure 2)
   Considerations before the reaction:
   Choosing an anaerobic cuvette. This method relies on strict anaerobic conditions required for the duration of the reaction. Some labs may be well equipped for this type of study and have a spectrophotometer placed in an anaerobic hood. For many labs, however, this option may not be available, thus the reaction should be well isolated from an aerobic environment by using specialized optical cuvettes (Figure 2). The classical anaerobic cuvette (Figure 2A) has a side arm and air is exchanged by repeated cycles of applying a vacuum and replacing air with nitrogen/argon gas. The xanthine oxidase (XO) which is used to initiate the reaction is placed in the side arm and mixed into the reaction after the cuvette is closed and sealed using vacuum grease. Alternatively, if the specialized cuvette shown in Figure 2A is unavailable, a standard stoppered cuvette (Figure 2B) can be used. Additions are made by a gas tight syringe through an oxygen impermeable septum (Figure 2B, top). The most convenient method which was used by us and others is to use a common stoppered cuvette (Figure 2B, bottom). To achieve anaerobic conditions required for the assay, we use the glucose/glucose oxidase/catalase oxygen scavenging system that efficiently eliminates dissolved oxygen. In addition, the cuvette head space is flushed with a low flow of argon/nitrogen. To initiate the reaction, XO is added with an air tight syringe and after the injection the reaction is carefully mixed with the syringe needle. For some assays when using very low potential flavoproteins or dyes (i.e., a potential less than -300 mV) the argon or nitrogen gas should be additionally treated by an oxygen scrubbing system to remove traces of oxygen. The use of an air tight cuvette (Figure 2A) would be also beneficial. The final volume of the reaction is usually 1 ml but can be scaled up or down depending on the cuvette type and amount of protein to be studied.
   
   
   Figure 2. Optical cuvettes that can be used for anaerobic measurements. A. A sealed anaerobic cuvette with a side arm. B. A cuvette with a septum (top) and a standard optical cuvette equipped with a silicone stopper (bottom). Both cuvettes can be used with a gas line to supply argon/nitrogen (shown for the bottom cuvette).
   
     Choosing an appropriate dye. As a general rule, the E_m of a reference dye should be within ±30 mV of the anticipated flavin potential in the protein of interest. Table 1 summarized some reference dyes that had been used for determination of flavin reduction potentials. Some tips for choosing a dye: for mutants of a flavoprotein with known potential start with a dye which has an E_m closest to the wild-type. For example, a mutation may result in a change of flavin attachment to the protein. Covalently bound flavins demonstrate higher reduction potentials than non-covalent flavins (Heuts et al., 2009). Since wild-type SdhA has a flavin potential of about -50 mV, we used a dye (ITS) which has a redox potential near this value (see Table 1).
   
   Table 1. Reference Dyes
   
   *Viologens are one-electron reduction dyes, all others undergo two-electron reduction.
   
     Choice of the wavelength to monitor absorbance changes in the flavin and dye. As depicted in Figures 1 and 4, the oxidized and reduced forms of the flavin and dye are calculated from the spectra containing both components. Thus, wavelengths used for the analysis should represent where spectral changes are attributed to only one component and where absorbance changes contributed by the second component are negligible. For example, the maximum absorbance of FAD in SdhA is around 460 nm, however, the reduced form of ITS also absorbs at this wavelength and will interfere with correct determination of FAD concentrations. A separate spectrum of dithionite reduced ITS, however, shows that 485 nm is the isosbestic point for ITS (demonstrated in Figure 3). FAD also retains significant absorbance at 485 nm, thus this wavelength can be used for calculations of reduced/oxidized forms of FAD without any contributions from ITS. Reduction of ITS was measured at 614 nm, a peak absorbance, where there is no interference from flavin absorption. Thus, the wavelengths of 485 nm for the flavin and 614 nm for the dye can be used for the analysis.
   
   
   Figure 3. The isobestic point for ITS. The spectrum of oxidized ITS is shown as the solid line and the dithionite reduced ITS is shown as the dashed line. Both forms show the same absorbance at 485 nm, i.e., the isobestic point.
   
   3.An air tight syringe for making additions to the anaerobic assay
   

Software

1. Microsoft Excel or any other spread sheet program for data analysis

Procedure