Scanning Optical Spectroelectrochemistry: Applications in Protein Redox Potential Measurements

Abstract

The accurate measurement of redox potentials of small molecules is a relatively straightforward task using electrochemical methods such as cyclic voltammetry. However, proteins, in most cases, are not amenable to the same approach due to slow heterogeneous electron transfer and the possibility of denaturing at the electrode surface. This necessitates the use of small molecular weight redox mediators to facilitate electron transfer. This leads to spectroelectrochemical techniques where the applied electrochemical potential is coupled to a spectroscopic signal of the protein. Traditionally this is done at different applied (fixed) potentials akin to an electrochemical titration, but the time required for electrochemical equilibrium to be established, and its consistent application, are major sources of experimental error. Here we have utilised a continuously scanning potential synchronised with time‐resolved UV‐vis spectroscopy to provide an automated approach that can be used to measure protein redox potentials accurately in an expedient manner. The test cases are the heme proteins cytochrome c and myoglobin. The scope and limitations of the method are discussed.

Redox Characterization of the Complex Molybdenum Enzyme Formate Dehydrogenase from Cupriavidus necator

“…The complexity of the optical spectroelectrochemistry of FdsDABG, with FMN and seven Fe/S clusters as chromophores (Figures S3 and S4), presents significant challenges in data analysis. Assuming that the absence of an EPR signal attributable to A3 (which would be similar to the signal of N4 in NADH dehydrogenase) is due to its low redox potential, we return to the low-potential region of the optical spectroelectrochemistry data collected on FdsDABG at high resolution (2.5 mV intervals, 520 spectra in all, Figure S4) in scanning mode 55 (Figure 15), and note that there is some evidence for two one-electron steps with redox potentials −0.44 and −0.51 V vs NHE (Figure 15B). Recall that in this region a clear reduction of B6 was seen in the FdsBG subcomplex, so one of these steps can be attributed to B6 in the FdsDABG holoenzyme.…”

“…(B) Absorbance at 440 nm as a function of potential and the calculated curve from the potentials [4Fe–4S] 2+/+ (1) −0.44 V and [4Fe–4S] 2+/+ (2) −0.51 V vs NHE which are indicated by the vertical lines. The hysteresis observed in the reductive and oxidative scans is due to mass transport limitations as described …”

“…A Gamry Interface 1010 potentiostat was used to provide potential control in either constant potential coulometry mode (with potentials measured at constant intervals) or in cyclic voltammetry mode (with the potential scanned slowly, as described). 55 In constant potential coulometry mode, the UV−vis spectra were taken when equilibrium was established and all absorbance changes ceased, which was usually 5 to 10 min. Spectra were taken at 12.5 to 50 mV intervals with reversibility established upon returning from low to high potential with little or no hysteresis.…”

“…UV–vis spectra were acquired with an Agilent 8453 diode array UV–vis spectrophotometer with the cell cooled at 15 °C with a Huber Ministat 230 Cooling Bath Circulation Thermostat. A Gamry Interface 1010 potentiostat was used to provide potential control in either constant potential coulometry mode (with potentials measured at constant intervals) or in cyclic voltammetry mode (with the potential scanned slowly, as described) . In constant potential coulometry mode, the UV–vis spectra were taken when equilibrium was established and all absorbance changes ceased, which was usually 5 to 10 min.…”

Electrochemical and structural characterization of recombinant respiratory proteins of the acidophilic iron oxidizer Ferrovum sp. PN-J47-F6 suggests adaptations to the acidic pH at protein level

“…The open circuit potential of the reaction mix was determined to set the initial potential for the reductive sweep. Before each potential sweep the initial potential was held for 120 s. Bernhardt, 2023 demonstrated the suitability of potential sweep rates between 0.1 and 0.6 mV/s for horse heart cytochrome c. Based on results on horse heart cytochrome c ( Bernhardt, 2023 ) using a similar experimental set-up, we ran reductive and oxidative potential sweeps in sequence between 450 mV and − 50 mV vs. Ag/AgCl at 0.1, 0.15, and 0.2 mV/s with a step size of 0.5 mV for each assay. UV/Vis spectra between 390 and 590 nm were recorded every 10 mV during the potential sweeps.…”