doi.org/10.26434/chemrxiv.7945379.v1

Electrochemically Gated Long Distance Charge Transport inPhotosystem IMontserrat López Martínez, Manuel López Ortiz, Maria Elena Antinori, Emilie Wientjes, Roberta Croce,Ismael Díez-Pérez, Pau Gorostiza

Submitted date: 03/04/2019 • Posted date: 04/04/2019Licence: CC BY-NC-ND 4.0Citation information: López Martínez, Montserrat; Ortiz, Manuel López; Antinori, Maria Elena; Wientjes, Emilie;Croce, Roberta; Díez-Pérez, Ismael; et al. (2019): Electrochemically Gated Long Distance Charge Transportin Photosystem I. ChemRxiv. Preprint.

The transport of electrons along photosynthetic and respiratory chains involves a series of enzymaticreactions that are coupled through redox mediators, including proteins and small molecules. The use of nativeand synthetic redox probes is key to understand charge transport mechanisms, and to design bioelectronicsensors and solar energy conversion devices. However, redox probes have limited tunability to exchangecharge at the desired electrochemical potentials (energy levels) and at different protein sites. Here, we takeadvantage of electrochemical scanning tunneling microscopy (ECSTM) to control the Fermi energy andnanometric position of the ECSTM probe in order to study electron transport in individual photosystem I (PSI)complexes. Current-distance measurements at different potentiostatic conditions indicate that PSI supportslong-distance transport that is electrochemically gated near the redox potential of P700, with currentextending farther under hole injection conditions.

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Electrochemically gated long distance charge transport in

photosystem I

Montserrat López-Martínez1,2,3,[+], Manuel López-Ortiz2,3, Maria Elena Antinori2,[++], Emilie

Wientjes4, Roberta Croce5, Ismael Díez-Pérez1,[+++], and Pau Gorostiza2,3,6,*

1Department of Material Science and Physical Chemistry, University of Barcelona, Martí i Franquès, 1 08028 Barcelona, Spain

2Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology Baldiri Reixac 10–12, 08028 Barcelona, Spain.

3Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Poeta Mariano Esquillor s/n, 50018 Zaragoza, Spain

4Laboratory of Biophysics ,Wageningen University, 6700 ET Wageningen, The Netherlands

5Biophysics of Photosynthesis. Dep. Physics and Astronomy, Faculty of Sciences, VU Amsterdam De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands

6Catalan Institution for Research and Advanced Studies (ICREA) Passeig Lluís Companys 23, 08010 Barcelona, Spain

*E-mail: pau@icrea.cat

[+]Present address: Institut für Angewandte Physik, TU Wien, Vienna, Austria

[++]Present address: Smart Materials, Nanophysics, Istituto Italiano di Tecnologia, Genova, Italy

[+++]Present address: Department of Chemistry, Faculty of Natural & Mathematical Sciences, King’s College London, London, UK

Abstract: The transport of electrons along photosynthetic and respiratory chains involves a series of enzymatic

reactions that are coupled through redox mediators, including proteins and small molecules. The use of native and

synthetic redox probes is key to understand charge transport mechanisms, and to design bioelectronic sensors and

solar energy conversion devices. However, redox probes have limited tunability to exchange charge at the desired

electrochemical potentials (energy levels) and at different protein sites. Here, we take advantage of electrochemical

scanning tunneling microscopy (ECSTM) to control the Fermi energy and nanometric position of the ECSTM probe

in order to study electron transport in individual photosystem I (PSI) complexes. Current-distance measurements

at different potentiostatic conditions indicate that PSI supports long-distance transport that is electrochemically

gated near the redox potential of P700, with current extending farther under hole injection conditions.

Photosynthesis is an essential process for plants, algae and bacteria since it converts light energy, CO2 and water

into carbohydrate molecules. Although the identity and structure of the main proteins and complexes involved in

photosynthesis have been extensively characterized,[1] the underlying electron transport pathways, their

mechanisms and regulation are not fully understood at the nanometer scale. Progress in this direction would also

enable the rational design of bioelectronic nanodevices based on photosynthetic complexes for solar energy

applications like hydrogen production[2] and photocurrent generation in photovoltaic cells.[3]

The photosystem I (PSI) complex is a light-driven oxidoreductase that accepts electrons from plastocyanin (Pc) or

cytochrome c6, and donates them to ferredoxin (Fd) or flavodoxin under native conditions. Among its ~200

cofactors, a special pair of chlorophylls (P700) located near the Pc binding site is known to photogenerate pairs of

electrons and holes that are subsequently transported in opposite directions across the thylakoid membrane. [4]

Biological and chemical redox mediators are key to investigate charge transport in these complexes, both for

fundamental and technological purposes.[5] P700 can accept electrons from a great variety of non-native donors

including organic redox compounds[6], organometallic complexes,[7] graphene,[8] and gold (Au) surfaces.[9] At the

opposite side of the complex, electrons can be withdrawn from the Fd binding site of PSI by non-native acceptors

like methylviologen[10] and can also be injected into the high energy conduction band of semiconductors like C60,[11]

TiO2 and ZnO.[12] Certain mediators like ferro/ferricyanide[13] or viologen derivatives[14] can act both as donor and

acceptor, showing that redox probes are useful at the macroscopic scale but provide very limited tunability to

exchange charge at the desired electrochemical potentials (energy levels) and different protein sites of the free

complex in solution. In addition, the distance dependence of charge exchange cannot be directly measured with

soluble mediators.[15]

As an alternative, electrochemical scanning tunneling microscopy (ECSTM) allows high-resolution imaging of

electrode surfaces, organic molecules and surface-immobilized proteins with a metallic nanoelectrode probe, whose

Fermi energy (potential) and nanometric position can be controlled independently and with high accuracy. [15, 16]

Here, we have used electrochemical tunneling spectroscopy (ECTS) to study for the first time electron transport in

individual PSI complexes in solution under bipotentiostatic control. In particular, we have taken advantage of the

probe adjustable potential and position to measure the distance dependence and the effect of the electrochemical

conditions, and we observe that PSI supports electrochemically gated long-distance charge transport.

We immobilized PSI isolated from plants[17] on the <111> surface of a gold monocrystal functionalized with 8-

mercaptooctanoic acid (8MOA) self-assembled monolayer (SAM) (Figure 1a). PSI has a disk-like structure with

polar stroma- and lumen-exposed sides and a hydrophobic edge that contacts the membrane. The charged regions

allow electrostatically immobilizing PSI onto SAMs with several terminal groups[18-21] using interactions resembling

those of PSI’s biological partners. In particular, negative SAMs like 8MOA have been shown to yield a high surface

coverage.[18-20] The macroscopic photo-electrochemical activity of PSI was preserved on these electrodes, as

shown by cyclic voltammetry (see the oxidation peak between 300-350mV vs Ag/AgCl in Figure 1b, in agreement

with previous reports[19, 21]) and amperometry (see the rapidly reversible photocurrent at 200 mV upon excitation of

P700 chromophores with 690 nm red light in Figure 1c[21]). ECSTM imaging showed PSI complexes homogeneously

distributed and individually resolved on SAM-functionalized Au<111> terraces (Figure 1d-e). Proteins display an

average diameter of 4.1 nm ± 1.0 nm and an apparent height of 0.31 nm ± 0.14 nm, which is lower than the

crystallographic height [22] but in the same range of previous reports of STM measurements in air.[23]

Figure 1. (a) Experimental setup for ECSTM imaging and I-z measurements of PSI on 8MOA-functionalized Au<111>

(sample working electrode: WE1; probe: WE2; Ag/AgCl reference: RE; counter: CE). (b) Cyclic voltammetry (10 mV/s)

of PSI on a MOA functionalized electrode (red) showing a peak near 400 mV, and CV for the same system in the

absence of PSI (grey). (c) Photocurrent in the presence (blue) and in the absence (black) of PSI at US = 200 mV under

intermittent illumination with red light (690 nm). Current traces were baseline subtracted for clarity. Red shadowed areas

correspond to ilumination periods. (d, e) ECSTM images of PSI at US=200 mV, Ubias= -300 mV, setpoint 0.5 nA.

We further investigated the distance dependence of the current between the ECSTM probe and PSI complexes.

The initial current setpoint was 0.5 nA in these current-distance (I-z) measurements, which were performed in six

electrochemical conditions (Figure 2): Ubias = 300 mV (probe potential UP = 500, 700, 900 mV; sample potential US

= 200, 400, 600 mV respectively, corresponding to electron collection at the probe), and Ubias = -300 mV (UP = -

100, 100, 300 mV; US = 200, 400, 600 mV respectively, corresponding to electron injection from the probe). Upon

disconnecting the feedback circuit, the probe was retracted at 8 nm/s while the current was recorded. The semi-

logarithmic I-z plots of Figure 2 display a nearly exponential current decay up to the faradaic current limit set by the

probe insulation (~1 pA). The exponential slope of each I-z curve yields a distance decay factor β (in nm-1) that can

be analyzed statistically in histograms for each electrochemical condition. Figure 2 contains more than 4000 I-z

plots obtained with five different probes and sample replicas. The ensemble of I-z recordings can be classified in 3

groups: (1) Plots showing an abrupt current decay (β > 8 nm-1) are similar to single-step tunneling between two

metals in vacuum or in electrolytic media, and are found in all sample potential and bias conditions in Figure 2, as

well as in all the control conditions (see Supporting Information). (2) Plots showing a gradual current decay (β ~ 4-

8 nm-1) are compatible with two-step tunneling observed by ECTS in other proteins[15]. Indeed, the reported

temperature independence of PSI currents[20] is in agreement with a tunneling based mechanism via multiple steps

through the long distances involved in the PSI pathway. (3) Plots displaying long current decay (β < 4 nm-1)

correspond to current recordings that extend beyond > 3 nm separation (indicated with stars in Figure 2). Charge

transport along such distances is not compatible with a tunneling mechanism but several interesting features are

observed. First, I-z plots with low β are found mostly at US ~ 200-400 mV (Figure 2), which is near the P700 redox

potential of PSI that is reported[19, 21] and observed in our voltammetry and amperometry recordings (Figure 1).

These low β values are also close to the redox midpoint of Pc, the native electron donor of PSI (170 mV [24]), in

agreement with results reported for other redox protein partners[25] (see further discussion below).

Figure 2. (a-f) I-z plots of PSI obtained at the indicated US and bias (0.5 nA initial setpoint; ensemble of 5 independent

experiments with different probes; number of curves: 1004 in a, 1002 in b, 1253 in c, 912 in d, 326 in e, 179 in f). Grey

curves correspond to the same experiments in the absence of PSI. Stars indicate traces with distance decay rates β below

4 nm-1 (g-l) Corresponding normalized distributions of distance decay rates (β) from the plots in (a-f). Arrows indicate

distributions of β values below 4 nm.-1

Electrochemical gating studies aim at measuring spectroscopic parameters (conductance[15, 26] and distance decay

rate[25]) as a function of the sample potential at constant bias potential. The results of Figure 2 show that long-

distance charge transport in PSI is electrochemically gated and yield low β values around the redox potential of

P700, suggesting that these effects are biologically relevant. In addition, the distribution of low β values strongly

depends on bias (Figure 3 displays I-z plots acquired in one experiment using the same ECSTM probe). An

increased occurrence of far-extending I-z plots, including decay rates as low as 1 nm-1, is observed at +300 mV

bias, which corresponds to electron withdrawal from the probe or “hole injection”. In the absence of illumination, this

condition could mimic hole photogeneration at P700 and provide an explanation of current directionality. The

interpretation that the P700 redox state controls a long-distance charge transport mechanism or pathway in PSI is

in full agreement with the function of the photosynthetic complex, and provides new fundamental insights on the

dark state of P700 that cannot be obtained using redox probes. Future ECTS studies include modifying the setup

to enable sample illumination in order to characterize the charge exchange with the photoexcited state P700*, and

the associated distance and potential dependences.

Figure 3. a) I-z plots of PSI at US = 200 mV and ±300 mV bias (0.5 nA initial setpoint, all recordings obtained with the

same probe; number of curves: 101 at -300 mV bias, 80 at 300 mV bias). Stars indicate traces with distance decay

rates β below 4 nm-1 (b) Corresponding distributions of β values. Arrows indicate distributions of β values below 4 nm-1.

Long-distance electron transport has been recently observed between redox partner proteins of the respiratory

chain.[25] As in the case of cytochromes c and bc1, electrostatic interactions play a key role in the binding between

PSI and its natural redox partners, Pc and Fd.[27] The PSI surface displays a heterogeneous electrostatic field,[27]

with two positively charged regions located around the binding sites of Pc and Fd. Both Fd and Pc bear a negatively

charged region around their binding sites with PSI that mediates the interaction between the partners. At low ionic

strength, charge screening is reduced and the electrostatic field over these regions becomes more pronounced,

extending several nanometers from the surface into the solvent.[28] This electrostatic distribution may create a

favorable conduit for charge transfer at long distance[25] , accounting for the low values observed in PSI (Figures

2 and 3). It has been suggested for different redox proteins that amino acid residues at the surface of the active site

rearrange the local solvent properties.[29, 30] This creates or stabilizes favorable paths for charge transport that are

mediated by water.[30, 31] The idea of an electrostatically stabilized water-mediated pathway is in agreement with the

recently reported structure of a redox protein pair interacting through three water layers. [32] Long-distance charge

transport between PSI and its partners would elude the requirements of a tightly bound complex, large

conformational rearrangements, and water reorganization, enabling in turn fast electron transfer rates.

We have shown that ECSTM allows studying charge transport through individual PSI complexes under

bipotentiostatic control, and reveals a long-distance current that is electrochemically gated around the redox

potential of P700. The current spatial extension is longer at bias corresponding to hole injection. These results are

important for understanding the basic principles of photosynthesis and have technological implications to electrically

control photosynthetic devices built on PSI and other protein-based nanobiodevices.

Acknowledgements

We acknowledge the help and advice of J. M. Artés, C. Orabona, F. Sanz, and N. van Hulst, and funding from the

BIST Ignite program, from the European Union (SGA 720270), Generalitat de Catalunya (CERCA and 2017-SGR-

1442), FEDER, and MINECO (CTQ2016-80066R and FPI fellowship BES-2013-066430 to M. L.-M.).

Keywords: electron transfer • photosynthesis • ECSTM • current decay • electrochemical gate

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Supporting Information

Electrochemically gated long distance electron transport in

photosystem I

Montserrat López-Martínez1,2,3,[+], Manuel López-Ortiz2,3, Maria Elena Antinori2,[++],

Emilie Wientjes4, Roberta Croce5, Ismael Díez-Pérez1,[+++], and Pau Gorostiza2,3,6,* 1Department of Material Science and Physical Chemistry, University of Barcelona, Martí i Franquès, 1 08028 Barcelona, Spain.

2Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology, Baldiri Reixac 10–12, 08028 Barcelona, Spain.

3Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Poeta Mariano Esquillor s/n, 50018 Zaragoza, Spain

4Laboratory of Biophysics ,Wageningen University, 6700 ET Wageningen, The Netherlands.

5Biophysics of Photosynthesis. Dep. Physics and Astronomy, Faculty of Sciences, VU Amsterdam De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands

6Catalan Institution for Research and Advanced Studies (ICREA) Passeig Lluís Companys 23, 08010 Barcelona, Spain.

*E-mail: pau@icrea.cat

[+]Present address: Institut für Angewandte Physik, TU Wien, Vienna, Austria.

[++]Present address: Smart Materials, Nanophysics, Istituto Italiano di Tecnologia, Genova, Italy.

[+++]Present address: Department of Chemistry, Faculty of Natural & Mathematical Sciences, King’s College London, London, UK.

Abstract: The transport of electrons along photosynthetic and respiratory chains involves a series of enzymatic reactions that are

coupled through redox mediators, including proteins and small molecules. The use of native and synthetic redox probes is key to

understand charge transport mechanisms, and to design bioelectronic sensors and solar energy conversion devices. However,

redox probes have limited tunability to exchange charge at the desired electrochemical potentials (energy levels) and at different

protein sites. Here, we take advantage of electrochemical scanning tunneling microscopy (ECSTM) to control the Fermi energy

and nanometric position of the ECSTM probe in order to study electron transport in individual photosystem I (PSI) complexes.

Current-distance measurements at different potentiostatic conditions indicate that PSI supports long-distance transport that is

electrochemically gated near the redox potential of P700, with current extending farther under hole injection conditions.

Table of Contents

Experimental Procedures 3

Supplementary Results 3

References 6

Experimental Procedures

Materials.

Chemicals where purchased from Sigma Aldrich. Solutions were prepared using Milli-Q water of 18 MΩ·cm, and were deoxygenated with N2. Glassware and electrochemical cells were cleaned with piranha solution 7:3 H2SO4/H2O2.

Electrode preparation.

Sample electrodes were Au<111> single crystals (Mateck) prepared as reported[1]. Briefly, they were annealed with butane flame for 3 minutes and cooled under argon stream. The crystal surface was electropolished in 0.1 M H2SO4 at 10 V for 30 s and the oxide was removed by two cycles of immersion in 1 M HCl for 2 min and Milli-Q water rinse. Finally, they were flame annealed again for 3 min and cooled with argon. After annealing, the sample surface was functionalized by immersion in 1 mM 8MOA in ethanol overnight and rinsed with ethanol and water. For voltammetric analysis, samples were functionalized for 48 h.

PSI purification and incubation.

PSI complexes were obtained and purified as reported[2]. The PSI solution was diluted (1:10) and the buffer exchanged by 50 mM phosphate buffered saline (PBS buffer) at pH 7.4 using Zeba Spin desalting columns (Thermo Scientific) in order to eliminate the excess of surfactant (dodecyl-β-maltoside, DDM) that could interfere with PSI adsorption. Au<111> samples were immediately incubated in 50 µl PSI-PBS solution for 2 h in the dark at room temperature.

Electrochemical measurements.

Voltammetric recordings were carried out in 50 mM PBS at pH 7.4 with a PGSTAT302N Autolab potentiostat (Metrohm-

Autolab) in three-electrode configuration with a low leakage reference electrode Ag/AgCl (World Precision Instruments)

and a Pt/Ir wire (Advent RM) as a counter electrode (CE). For the photocurrent measurements, sample potential was kept

at US = 200 mV. Illumination with a red LED (Kingbright Electronic, 690 nm) built in the cell was switched on and off

manually every 10 s.

ECSTM and ECTS.

Imaging and spectroscopy recordings (ECTS) were carried out with a PicoSPM microscope head and a PicoStat bipotentiostat (Molecular Imaging) controlled by Dulcinea electronics (Nanotec Electronica). A homemade electrochemical cell was used in four-electrode configuration, with a Pt/Ir (80:20) wire counter electrode and a miniaturized ultralow leakage membrane Ag/AgCl reference electrode filled with 3 M KCl (World Precision Instruments, Sarasota, FL). The potentials of the Au<111> sample (US) and apiezon-insulated Pt/Ir ECSTM probe[3] (UP) are expressed against this reference. The recording solution was 50 mM PBS buffer at pH 7.4. Images were analyzed with WsxM 4.0 Software[4] and with a home

written program to identify and measure the proteins. The program is based on Adam Ginsburg’s gaussfitter [5] and available at (https://github.com/Mlopeorti/Blob-2D-fitter-). Iz curves were analyzed using Origin and a home written programm available at (https://github.com/Mlopeorti/Tunnel_current_2exponetial_fitter). I-z curves were recorded at different UP and US potentials using an initial setpoint of 0.5 nA. Upon stabilization, I-z recordings were launched by interrupting the feedback and retracting the probe along 5 nm at 8 nm/s. The probe was reapproached to the surface and the feedback resumed. I-z recordings were acquired at multiple locations on the sample surface. To minimize drift artifacts, I-z plots that started with a current deviation higher than 10% of the setpoint were discarded[3]. ECTS spectra were fit

individually to 𝐼(𝑧) = 𝐼0𝑒−𝛽𝑧 to obtain the distance decay factor β[6]. Spectra with r2 < 0.95 were discarded for the analysis.

Supplementary Results

Figure S1 shows the ECSTM images of (a-b) PSI directly bound to an Au<111> electrode without SAM (c) DDM surfactant incubated on the same surface.

Figure S1..a and b) ECSTM images of PSI on a Au electrode, with US=200 mV and Ubias= -300 mV. The current setpoint was 0.5 nA. c) ECSTM images of DDM detergent on a Au electrode, with US=200 mV and Ubias= -300 mV. The current setpoint was 0.5 nA. All potentials are expressed against Ag/AgCl reference electrode.

Figure S2 shows the semilogaritmic I-z plots of the control curves shown in Figure 2 in the absence of PSI, where we observe a range of spectra with a broad distribution of β values centered around ~ 8 nm -1 with no significant dependence on the electrochemical potential. Curves for the different components (Au, detergent) were analyzed (figure S3, S4, S5) obtaining equivalent results. Although the distribution of values is quite broad, low β values (below 4 nm -1) were only observed for PSI (Figure S6).

Figure S2. Left curves: I-z curves for 8-MOA functionalized gold electrodes (raw data) obtained for different sample potentials (US = 200 mV, 400 mV, 600 mV, from top to bottom) and -300 mV (left) and +300 mV (right) bias. Right histograms: Statistical representation of the β values obtained for the shown curves. The current setpoint was always 0.5 nA.

Figure S3. Left curves: I-z curves for clean gold electrodes (raw data) obtained for different sample potentials (US = 200 mV, 400 mV, 600 mV, from top to bottom) and -300 mV (left) and +300 mV (right) bias. Right histograms: Statistical representation of the β values obtained for the shown curves. The current setpoint was always 0.5 nA.

Figure S4. Left curves: I-z curves DDM surfactant incubated on clean gold electrodes (raw data) obtained for different sample potentials (US = 200 mV, 400 mV, 600 mV, from top to bottom) and -300 mV (left) and +300 mV (right) bias. Right histograms: Statistical representation of the β values obtained for the shown curves. The current setpoint was always 0.5 nA.

Figure S5. Superimposed histograms corresponding to all controls in the absence of PSI.

Figure S6. Distribution of β values for a bare gold electrode, DDM surfactant incubated on a clean gold electrode, and 8-mercaptooctanoic acid

SAM on a gold electrode, does not show any counts for β<4. The distribution for PSI β values β<4 is shown for comparison

References

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