Long distance electron transfer through the aqueous solution between redox partner proteins

Despite the importance of electron transfer between redox proteins in photosynthesis and respiration, the inter-protein electron transfer rate between redox partner proteins has never been measured as a function of their separation in aqueous solution. Here, we use electrochemical tunneling spectroscopy to show that the current between two protein partners decays along more than 10 nm in the solution. Molecular dynamics simulations reveal a reduced ionic density and extended electric field in the volume confined between the proteins. The distance-decay factor and the calculated local barrier for electron transfer are regulated by the electrochemical potential applied to the proteins. Redox partners could use electrochemically gated, long distance electron transfer through the solution in order to conciliate high specificity with weak binding, thus keeping high turnover rates in the crowded environment of cells.


Estimation of the extended protein length
The length of the extended proteins can be estimated from the corresponding amino acid (AA) sequences and assuming an averaged AA length of 0. I-z curves were recorded at different probe (U P ) and substrate (U S ) potentials, using a constant bias potential (U bias = U P -U S ). The potential of the reference electrode is adjusted to modulate the gate voltage in analogy to the gate electrode in a field effect transistor (FET). As the ECTS probe is grounded in our STM electronic configuration, the EC gate corresponds to -U S at any given bias. 29

Supplementary Figure 1
The electrochemical potentials applied to the probe (hCc) and the sample (pCc 1 ) in the ECSTM set-up mimic the sense of the physiological electron flow. Schematic representation of the charge flow (depicted with arrows) through complex II (CII) and respirasome, which comprises complexes I (CI), III (CIII) and IV (CIV) in the mitochondrial respiratory chain. Red arrows indicate the sense of the electron flow from the ubiquinone (Q) pool, through CIII, cytochrome c (Cc) and CIV to oxygen. The electron flow is associated with proton extrusion to the mitochondrial intermembrane space, which produces the electrochemical gradient used in ATP synthesis. ECTS experiments were performed at electrochemical potentials in which pCc 1 on the substrate is reduced (U S < Em pCc1 ) and hCc in the ECSTM probe is oxidized (U P > Em hCc ), resulting in a constant positive bias. Under these conditions, electrons are transferred from pCc 1 to hCc in analogy to physiological ET.

Supplementary Figure 2
Current-distance spectroscopy of specific ET between redox partners hCc (probe) and pCc 1 (sample), compared to the non-specific interaction between hCc in both probe and sample electrodes (self-ET). a, Ensemble of semi logarithmic retrace I-z plots showing that the ET current decay with the distance is more pronounced for hCc-hCc (self-ET; blue) than for pCc 1 -hCc (red). b, Histograms of distance-decay constant β quantified from individual curves in a. Significant differences (P < 0.01; non-parametric Kolmogorov-Smirnov test) were found between pCc 1 -hCc and hCc-hCc β distributions (red and blue histograms respectively), indicating that ECTS is sensitive to the specificity between the redox partner proteins (see also Fig.1cd and Suppl. Fig 3). Experiments were performed at U S = -200 mV and U P = 600 mV in 50 mM sodium phosphate buffer, pH = 6.5. Initial set point 0.4 nA.

Supplementary Figure 3
Current-distance ECTS of the interaction of pCc1 or hCc with bare gold. a, Ensemble of semi logarithmic retrace I-z plots recorded for pCc 1 (green), and for hCc-coated gold (orange). b, Corresponding histograms of β quantified from individual curves in a. Dashed lines are an eye guide. β values (given as the mean with its standard error) are twice as high as the values obtained for the specific pCc 1 -hCc interaction (Fig. 1), indicating that ECTS is sensitive to the presence of proteins in both electrodes. Experiments were performed at U S = -200 mV and U P = 600 mV in 50 mM sodium phosphate buffer, pH = 6.5. Initial set point 0.4 nA.

Supplementary Figure 4
Long distance ET between pCc 1 -hCc is driven by the electrical double layer at the interface. The β values obtained experimentally for bare gold (grey) and for pCc 1 -hCc (red) are correlated with the calculated inverse Debye-Hückel length (К -1 ) at the same ionic concentration. It is observed that at low ionic concentration (low К -1 ) the spatial range of ET is higher (the distance decay constant β is lower). The numerical correlation between β and 1/К -1 for pCc 1 -hCc indicates that the current and potential between proteins are driven by similar distance-decay lengths.

Supplementary Figure 10.
Distance decay factor (β) and local barrier height (Φ local ) reach minima near the redox potentials of pCc 1 -hCc in electrochemically gated long distance ET. a, Cyclic voltammetry of Au(111) electrodes modified with hCc (left) and pCc 1 (right). The corresponding reduction (U c ) an oxidation (U a ) peaks for the Fe 3+ /Fe 2+ couple are indicated. b, Representative semi-logarithmic I-z plots obtained at 200 mV constant bias for pCc 1 -hCc at the indicated probe (U P ) and sample (U S ) potentials (upper row) and the corresponding Φ-z plots calculated according to ref. 27 (lower row). Near U S = 0.25 V/SSC the current decrease is more gradual and β and Φ local are lower than at other