The overall goal of this protocol is to probe the active side chemistry of a nickel-iron hydrogenase redox enzyme under both non-turnover and steady-state electrocatalytic turnover conditions using protein film infrared electrochemistry or PFIRE. The main advantage of the PFIRE technique is that it enables simultaneously precise electrochemical control and infrared spectroscopic sampling of redox proteins immobilized on a carbon electrode. This method can help answer key questions in the fields of biophysics and bioelectrochemistry about which states of redox protein are present during steady-state catalytic turnover.

To begin the procedure, in a wet anaerobic glove box suspend 20 milligrams of high surface area carbon black particles in one milliliter of ultrapure water. Sonicate the suspension at lower power for at least 15 minutes or until the particles are uniformly dispersed and no sediment forms within one hour at rest. Then load 15 microliters of an approximately seven milligram per milliliter solution of E.Coli hydrogenase one into a 50 kilodalton centrifugal filter unit.

Dilute the solution with 450 microliters of a low ionic strength exchange buffer with a pH close to the isoelectric point of the hydrogenase. Concentrate the mixture to 50 microliters by centrifugation at 27, 000 times g. Reconcentrate the mixture four more times to complete the buffer exchange.

Then combine five microliters of the 20 milligram per milliliter carbon black dispersion with the buffer exchanged hydrogenase. Store the mixture at zero degrees Celsius overnight to allow the hydrogenase to absorb to the carbon black particles. Periodically check the mixture to maintain the particle dispersion.

Centrifuge the modified particles at 27, 000 times g and verify that the supernatant is nearly colorless, indicating good absorption of the hydrogenase to the particles. Achieving a high level of absorption is critical for the success of the experiment. We optimize the absorption buffer using the low ionic strength buffer at the pH close to the isoelectric point of the protein as a starting point.

Wash the particles with three to five cycles of centrifugation and resuspension in fresh exchange buffer. Concentrate the particle mixture to approximately five microliters to achieve a 20 milligram per milliliter particle loading. To begin preparing to take PFIRE measurements, clean a silicone internal reflection element by low power sonication in sulfuric acid for 15 minutes.

Followed by nitric acid for one hour. Then rinse the element in ultrapure water and dry it under a stream of dry nitrogen gas. Use electrical grade silicone sealant to fix the IRE into the baseplate of a five reflection ATR accessory taking care to keep the sealant to edges of the IRE.

Allow the sealant to dry completely. Then bring the baseplate into a dry anaerobic glove box with an IR transparent window next to an FTIR spectrophotometer. Mount the baseplate on the ATR accessory.

Acquire a background spectrum in rapid scan mode. Then detach the ATR accessory baseplate and transfer it to the wet anaerobic glove box. Dropcast one microliter of the enzyme-modified carbon black particles evenly over the IRE surface without allowing the particles to dry completely.

It is important that the enzyme-modified particle mixture has been concentrated to a loading as close to 20 milligrams per milliliter as possible. Otherwise, it can be difficult to achieve a well-connected particle film when dropcasting the particles in this step. Gently place a piece of carbon paper soaked in ultrapure water onto the IRE surface, ensuring that the particle film is covered without allowing the paper to contact the silicone sealant.

Mount a custom spectroelectrochemical cell over the IRE. Add 200 microliters of experiment buffer via the solution inlet to keep the enzyme hydrated during the system preparation. Connect the solution inlet and outlet to a vial of experiment buffer via peristaltic pump tubing.

Then transfer the assembled cell to the dry glove box. Mount the cell assembly on the ATR accessory and connect the tubing to the peristaltic pump. Acquire an absorbent spectrum using the previously acquired spectrum as the background.

Verify that the MI2 bands are strongly visible at 1, 540 reciprocal centimeters and that the hydrogenase active site peaks are detectable in the 1, 850 to 2, 150 region. To prepare for the experiment, apply a reducing potential of negative 0.8 volts versus a saturated calomel reference electrode to the particle film. Saturate the experiment buffer with anaerobic hydrogen gas.

Then begin flowing the buffer through the spectroelectrochemical cell at about 12 milliliters per minute. Leave the sample under the flow of hydrogen-saturated experiment buffer overnight to activate the hydrogenase. Acquire an absorbent spectrum of the activated sample and verify that the CO and CN bands of the active site show multiple reduced states.

Next saturate the experiment buffer with anaerobic nitrogen gas and flow the buffer through the cell. Apply an oxidizing potential of zero volts versus a saturated calomel reference electrode for 30 minutes and acquire an absorbent spectrum. Then apply a reducing potential for 30 minutes and acquire another spectrum.

Verify that the enzyme was completely oxidized and then reduced. If not, check the electrical connections of the cell. Using anaerobic hydrogen saturated buffer, acquire a series of cyclic voltammograms at increasing flow rates to determine the optimal flow rate for the experiment.

Using this flow rate, acquire spectra at a range of potentials and solution conditions. PFIRE measurements of E.coli hydrogenase one were obtained at various potentials in an inert atmosphere and in the presence of hydrogen gas. The spectra acquired under a hydrogen atmosphere represented the steady-state distributions of the active site states present during catalytic hydrogen oxidation.

The anaerobic oxidative and activation of the hydrogenase via formation of nickel-B state from the nickel-Si was then investigated by acquiring spectra at various time points during potential application and preparing different spectra relative to the first spectrum. The observed gradual conversion of nickel-Si to nickel-B was consistent with the monotonic decrease in the current. Spectra were also acquired over a range of solution pH to investigate the proton transfer steps of the hydrogenase catalytic cycle.

At low pH, the nickel-C state was more prevalent whereas the nickel-L state was more prevalent at high pH. The pH dependence of the relative concentrations of nickel-C and nickel-L was determined from the maximum absorbents values at the respective peaks at each pH evaluated in the experiment. This technique paves the way for researchers in the field of bioelectrochemistry to explore steady-state kinetics of hydrogen activation by hydrogenesis.

After watching this video, you should have a good understanding of a typical PFIRE experiment. The technique is suitable for any redox protein that can be studied by protein film electrochemistry and adds direct chemical insight to the electrochemical measurement.