3.6.1. Amidation of the Thioctic Acid With Nitriolacetic Acid
Starting from the SAM configuration described in Subheading 3.4., SAMs of dithioctic acid activated with the NHS, the electrode is modified with A/-(5-amino-1-carboxypentyl)iminodiacetic (ANTA), a compound that forms stable octahedral complex with Cu(II), Ni(II), Co (II) and Zn(II), while leaving two vacant sites for coordination of nucleophiles like histidines.
1. Incubate overnight the gold covered with a SAM-TOA-NHS in a 0.15 Msolution of ANTA in DMF/H2O (2:1). Alternatively, a mixture of ANTA and EA can be used in a molar ratio range from 1 to 0.1 (total concentration of amine reactive group, 0.15 M) to prepare electrodes where the ANTA ligands were diluted with shorter hydroxy-ending molecules.
2. Wash the electrodes with DMF.
For a copper chelate, this step consists on dipping the modified electrode in a 40 mM CuSO4 solution, pH 5.5, (acetate buffer 50 mM) for 1 h. If cobalt is desired, work the same way with 40 mM CoCl2 salt instead of the copper one. All solutions must be prepared with Millipore (Milli-Q plus) water (18.2 MQ • cm).
3.6.3. Immobilization of His-Tagged Ferredoxin-NADP Reductase
1. Incubate the gold wire with its surface covered by SAM-TOA-NTA-Cu2+ in 50 mM phosphate buffer solution containing 5 mM imidazole and 1.4 ||M of FNR mutant with exposed histidine pairs.
2. Wash the electrodes with 50 mMphosphate buffer and 0.15 MKCl, pH 7.5.
The enzyme ferredoxin-NADP+ reductase catalyzes the oxidation of NADPH with artificial electron acceptors (diaphorase activity [21J). Therefore, the course of the enzymatic reaction was followed by CV analysis of the reaction product, using the same methodology described in Subheading 3.5.4. above.
1. Insert the FNR-SAM-modified gold wire in a conventional electrochemical cell with 2 mL of 50 mM phoshate buffer, pH 7.5, 0.1 MKCl, and 20 |Mferrocenemethanol. Insert a platinum auxiliary electrode and a Ag/AgCl, 3 M NaCl calomel electrode, connected to the cell by a salt bridge filled with the electrolyte solution.
2. Connect the cell to a electrochemical analyzer such as the CV50, BAS analyzer.
3. Add the substrate NADPH to a final concentration of 2 mM and immediatedly start to record b bcyclic voltammograms at 10 mV/s . The current-potential curve shows a plateau.
4. Wash the cell and repeat steps 1-4 in the absence of NADPH. Subtract the plateau current from the current measured in step 4. The net current is proportional to the activity of the enzyme in the electrode.
5. Repeat steps 1-5 at different concentration of substrates to estimate the kinetic constants according to the methodology described by Bourdillon et al. (22) (see Note 2).
3.6.5. Reversibilityofthe Chimeric Protein Binding
Incubation of the protein monolayer in 100 mMimidazol solution to release the FNR from the electrode.
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