Pyruvate:ferredoxin Oxidoreductase (PFR1) Activity Assays Using Methyl Viologen as Artificial Electron Acceptor

Figure 1. Activity assay using pyruvate, coenzyme A and methyl viologen (Ctrl+). In the absence of pyruvate, no methyl viologen reduction occurs (Ctr-).

Materials and Reagents

         Note: All Reagents are dissolved freshly in an anaerobic tent.

1. Purified pyruvate: ferredoxin oxidoreductase (PFR1)
   
2. Sodium pyruvate (≥ 99%, Stock 500 mM) (Sigma-Aldrich)
   
3. Sodium coenzyme A (≥ 85%, Stock 20 mM) (Sigma-Aldrich)
   
4. Thiamine pyrophosphate (≥ 95%, Stock 250 mM) (Sigma-Aldrich)
   
5. Methyl viologen (98%, Stock 1 M) (Sigma-Aldrich)
   
6. Dithioerythriol (≥ 99%, Stock 100 mM) (Carl Roth)
   
7. 0.1 M Tris-HCl buffer (pH 8.0) (see Recipes)
   

Equipment

1. Anaerobic tent (1% H₂, 99% N₂) (Toepffer Lab Systems)
   
2. 96 well plate reader (Beckman Coulter, catalog number: Paradigm1113 )
   
3. PC running Multimode analysis software (Beckman Coulter)
   
4. NanoDrop (Paqlab)
   

Procedure

1. Protein concentration of heterologous expressed and purified PFR1 from 2 L of cell culture is measured at A_280nm using NanoDrop. (ref bio-protocol: Heterologous Production and Anaerobic Purification of His- and StrepII-tagged Recombinant Proteins).
   
2. All reduction assays are performed under anaerobic atmosphere (1% H₂, 99% N₂) at room temperature.
   
3. The reaction mixture contains 10 mM sodium pyruvate, 2 mM sodium coenzyme A, 5 mM thiamine pyrophosphate, 10 mM methyl viologen and 16 mM dithioerythritol in 0.1 mM Tris-HCl (pH 8).
   
4. To start catalysis a final concentration of 1.4 μM PFR1 is added to the reaction mixture and absorbance (A₆₀₄, 96 well plate reader) can be monitored time resolved every 30 seconds until saturation is reached.
   
5. To determine enzyme activity the molar extinction coefficient ε₆₀₄ = 13.6 mM^-1 cm^-1 (Mayhew, 1978) can be used applying Lambert Beer Law (Eq.I). One unit was defined as the conversion of 1 mol of pyruvate or CoA and the reduction of 2 mol of methyl viologen, respectively, per minute.
   
   E_λ = ε_λ ^. c ^. d                 (Eq.I)
   E_λ: extinction
   ε_λ: extinction coefficient
   c: concentration
   d: layer thickness
   

Recipes

1. 0.1 M Tris-HCl buffer (pH 8.0) (1,000 ml)
   Mix 12.114 g of Tris base with 800 ml dH₂O
   Adjust pH to 8 with HCl
   Add ddH₂O to 1,000 ml
   Autoclave for 20 minutes at 121 °C
   Store at 4 °C
   

Acknowledgments

Kinetics for enzyme dependent methyl viologen reduction is adapted from Zeikus et al. (1977). Research on the pyruvate:ferredoxin oxidoreductase from C. reinhardtii was scientifically supported by Anja Hemschemeier and Thomas Happe.

References

1. Mayhew, S. G. (1978). The redox potential of dithionite and SO-2 from equilibrium reactions with flavodoxins, methyl viologen and hydrogen plus hydrogenase. Eur J Biochem 85(2): 535-547.
   
2. Noth, J., Krawietz, D., Hemschemeier, A. and Happe, T. (2013). Pyruvate:ferredoxin oxidoreductase is coupled to light-independent hydrogen production in Chlamydomonas reinhardtii. J Biol Chem 288(6): 4368-4377.
3. Noth, J. (2013). Heterologous production and anaerobic purification of His- and StrepII-tagged recombinant proteins. Bio-protocol 3(17): e881.