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. 2025 Oct;21(42):e01286.
doi: 10.1002/smll.202501286. Epub 2025 Sep 12.

Long-Distance Charge Transport between Cytochrome c and Complex III is Mediated by Protons and Reactive Oxygen Species

Anna Lagunas  1   2 , Alexandre M J Gomila  1   3 , Alba Nin-Hill  4 , Alejandra Guerra-Castellano  5 , Gonzalo Pérez-Mejías  5 , Josep Samitier  1   2   6 , Carme Rovira  4   7 , Miguel A De la Rosa  5 , Irene Díaz-Moreno  5 , Pau Gorostiza  1   3   7
Affiliations

Long-Distance Charge Transport between Cytochrome c and Complex III is Mediated by Protons and Reactive Oxygen Species

Anna Lagunas et al. Small. 2025 Oct.

Abstract

Electron transfer (ET) between redox proteins is an essential process in the respiratory and photosynthetic transport chains. While intra-protein ET is well characterized, the experimental methods to investigate inter-protein ET are limited by the presence of the solvent and by the transient nature of the protein-protein interaction and ET event, which are averaged in protein ensembles. Wiring precisely oriented redox protein partners to the nanoscale electrodes of an electrochemical scanning tunneling microscope allows recording the time- and distance-dependence of the current flowing between them. These methods have revealed that the current flowing between individual protein pairs extends beyond tunneling distances and that it is electrochemically gated. However, the corresponding mechanism and the identity of the charge carriers in aqueous solution remain to be elucidated. To determine the species involved in long-distance charge transport between the redox partner proteins Cc and Cc1 of the respiratory chain, recordings are performed as a function of pH, in heavy water solutions, and in degassed solutions. It is observed that the spatial span and electrochemical gating of long-distance currents are reduced at high pH, in heavy water, and at low oxygen concentration, showing that the currents are assisted by superoxide anions and by protons.

Keywords: Gouy‐Chapman conduit; Grotthuss (Grothuss) proton hopping conduction; electrochemical STM; kinetic isotope effect KIE; mitochondria; proton coupled electron transfer PCET; reactive oxygen species ROS; superoxide radical anion SOX.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effect of proton concentration in the current‐distance decay between Cc 1‐Cc. a) Scheme of the current‐distance (I‐z) measurement between Cc and Cc 1 with EC‐STM showing Cc (orange) bound to the EC‐STM probe and Cc 1 (green) bound to the surface of Au(111) electrode. b) Ensemble of semi‐logarithmic I‐z curves (left) obtained in 50 mm phosphate buffer at pH 6.6 (red), pH 7.4 (blue), and pH 8.0 (purple), and the corresponding histograms of distance decay factors (β) quantified from individual curves (right). c) Comparison of the averaged β values for Cc‐Cc 1, β = 0.6 ± 0.3 nm−1 at pH = 6.6 (n = 123 curves, N = 2, red) and β = 1.5 ± 0.4 nm−1 at pH = 7.4 (n = 109 curves, N = 2, blue), respectively, showing significant differences (Mann Whitney test, U = 748, two‐tailed, ****p < 0.0001).
Figure 2
Figure 2
Current‐distance electrochemical tunneling spectroscopy of Cc‐Cc 1 in deuterated water (D2O). a) (Left) Ensemble of semi‐logarithmic I‐z curves obtained for Cc‐Cc 1 in 50 mm phosphate D2O buffer solution of pD = 6.9 (green), and in 50 mm phosphate buffer aqueous solution pH 6.6 (red). Sample and probe electrodes are represented by a square and a triangle with bound Cc 1 and Cc, respectively. (Right) The corresponding β histograms quantified from individual I‐z curves. b) Comparison of β distributions (one‐way ANOVA with Tukey's multiple comparisons post hoc test, α = 0.05, *p < 0.05; ****p < 0.0001). c) Averaged β values (mean ± s.d. of nH2O at least 57, nD2O at least 61, N = 2) versus the electrochemical gate potential.[ 34 ] The gray area corresponds to the region comprised between the midpoint redox potentials of Cc 1 at the sample (0.28 V vs SSC) and of Cc at the probe (0.35 V vs SSC), respectively.
Figure 3
Figure 3
Current‐time measurements in water and deuterated water. a) Scheme of the current‐time (I‐t) measurement between Cc and Cc 1 in the EC‐STM showing Cc (orange) bound to the EC‐STM probe and Cc 1 (green) bound to the Au(111) electrode surface. b) Representative I‐t recording for Cc‐Cc 1 in 50 mm phosphate D2O buffer solution of pD 6.9 (green), and in 50 mm phosphate buffer aqueous solution pH 6.6 (red). c) 2D histograms of blink current and blink lifetime (nD2O = 166 095 and nH2O = 100 980 conductance values included). Counts are normalized and represented on a color scale. G0 = 2e 2/h = 77.5 µS, with h the Planck constant and e the electron charge.
Figure 4
Figure 4
Residues displaying redox‐dependent pK a in Cc. Front view of the Cc active site displaying the heme group in yellow. The pK a values of each residue were calculated using H++ in reduced (a) and oxidized (b) Cc structures[ 48 ] and represented using a color code (see Spreadsheet S1, Supporting Information). Twelve residues out of 103 display a difference in pK a higher than one pH unit between the reduced and oxidized forms (see Spreadsheet S1, Supporting Information). Four of them agree with the predictions of PropKa3.5 (green in panel c). Since all Cc residues are at tunneling distance from the heme site, this network of redox‐dependent (de)protonating sites can switch its availability to form hydrogen bonds with water and/or to mediate PCET reactions, leading to long‐distance charge transport between the redox partners along the extended electric field of the GCC.
Figure 5
Figure 5
Effect of O2 concentration in the current‐distance decay between Cc 1‐Cc. Ensemble of semi‐logarithmic I‐z curves (left), and the corresponding β histograms (right) of Cc‐Cc 1 in 50 mm phosphate buffer at pH 6.6, obtained during the first 10 min of recording (grey, n = 119, N = 2) and after 10 min (black, n = 130, N = 2). The buffer was degassed with nitrogen for >1 h before the measurements, and the experiment was conducted under argon atmosphere in the closed EC‐STM chamber (non‐hermetic). Comparison of the β distributions leads to statistically significant differences (Mann–Whitney test, U = 5400, two‐tailed, ****p < 0.0001).
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