Electrocatalytic Currents from Single Enzyme Molecules

Transcription

1 Supporting Information Electrocatalytic Currents from Single Enzyme Molecules Alina N. Sekretaryova, * Mikhail Yu. Vagin,, Anthony P.F. Turner, and Mats Eriksson Department of Physics, Chemistry and Biology, Linköping University, SE , Linköping, Sweden Department of Science and Technology, Linko ping University, SE , Norrko ping, Sweden *Corresponding author Alina N. Sekretaryova alina.sekretareva@liu.se Phone: +46(0) S-1

2 EXPERIMENTAL DETAILS Laccase activity assay. Laccase from Trametes versicolor (molecular mass of 70 kda, activity 10 U mg -1 (Sigma, Sweden)) was used in all experiments. The enzyme was used as received without further purification. One unit of laccase activity was defined as the amount of enzyme oxidizing 1 μmol substrate per minute. Suitable amounts of enzyme necessary to obtain an extinction of after approximately 1.5 min were used in photometric measurements. The 2,2'- azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) ABTS (ε 420 = M -1 cm -1 ) test was performed in 0.1 M acetate buffer at ph 5.0 with 0.8 mm final concentration of the substrate. 1 The laccase activity was found to be 10.5 ± 0.4 U mg -1. Dynamic light scattering measurements. The size of enzyme molecules and the zeta potential of the molecules in 0.1 M acetate buffer (ph 5.0) were determined using a Nano ZS dynamic lightscattering Zeta potential instrument (Malvern Instruments, Worcestershire, UK). Electrochemical measurements. All electrochemical experiments were performed using an Ivium Stat.XR electrochemical analyzer (Eindhoven, Netherlands) with a three-electrode cell placed in a Faraday cage covered inside and outside by an additional protective layer to reduce acoustic noise. A Pt wire was used as a counter electrode and the reference electrode was Ag/AgCl (3 M KCl). Gold microdisk electrodes (AuUMEs) with a diameter of 12.5 μm (CHI105, CH Instruments) were used as working electrodes. Before each use, the AuUMEs were polished with 0.05 μm Al 2 O 3 powder on microcloth pads (Buehler, Lake Bluff, IL) and rinsed thoroughly with Mili-Q water. The reproducibility of the surface was checked in a solution of the standard redox probe 1,1'- ferrocenedimethanol (Figure S6). Recording and analyzing single enzyme molecule collisions. Measurements were performed with Ag/AgCl as a reference electrode and then rescaled to yield potential vs the normal hydrogen electrode (NHE). Current transients were recorded at a data acquisition rate of 1-10 ms with noise filtering at 10 Hz. Before injection of enzyme solution, the AuUME was held at an applied potential until a steady-state current was achieved. Measurements were paused when the Faraday cage was opened for enzyme and inhibitor solution injections, in order to reduce the noise that would otherwise appear. Impact spikes were analyzed using the program Origin v.8.0 ( for spike identification and integration. Electrical noise was removed by applying Fourier transform filtering (within Origin software) at 10 Hz. Spikes were automatically identified by the same software at a threshold of 10 % of the highest spike. S-2

3 Figure S1. Monitoring the electrocatalytic current from individual enzyme molecules. Amperometric i-t curves in the presence of 0.1 U ml -1 enzyme solution in an oxygen containing solution. The AuUME was biased at V vs NHE over the entire experimental time. Experimental conditions were: 0.1 M acetate buffer, T=20 C. The varying noise between the curves is related to irreproducibility of the AuUME surface roughness between the experiments, introduced by polishing, and to variations of the external influences. S-3

4 Supporting Note 1. Calculation of the mass-transfer limiting current of an enzyme molecule. The amplitude of a current spike for a mass-transfer limiting current generated at an individual spherical catalytic nanoparticle in contact with the electrode can be estimated by: 2 I = 4π(ln2)nFDCr (1) where n is the number of electrons transferred in the reaction, F is the Faraday constant, D is the diffusion coefficient of the reactant at concentration C and r is the radius of the nanoparticle. In our case n = 4, D of oxygen in water according to literature 3 is cm 2 s -1, the concentration of dissolved oxygen in water 4 is 9.09 mg dm -3 ( mol cm -3 ) and the radius of the enzyme globule is ~3 nm ( cm) (according to literature 5 and from the dynamic light scattering experiment). Therefore, according to eq. (1) the value of the peak current amplitude should be of the order of 5.6 pa assuming the whole enzyme molecule is catalytically active. A smaller value of the observed peak current amplitude is expected, however, since only part of the enzyme molecule is catalytically active and due to restrictions of the oxygen diffusion. S-4

5 Figure S2. Influence of inhibition on the observed catalytic currents from single enzyme molecule collisions. Amperometric i-t curve in presence of 0.1 U ml -1 enzyme solution in oxygen containing solution before and after addition of an inhibitor, NaF solution, to a final concentration of 100 mm. The AuUME was biased at V vs NHE over the entire experimental time. Experimental conditions were: ph 5.0 (0.1 M acetate buffer), T=20 C. S-5

6 Figure S3. Dynamic light scattering measurements of enzyme molecule size. 10 U ml -1 enzyme solution in 0.1 M acetate buffer (ph 5.0), T=20 C The solid line is a curve fit to the experimental data. The measurement indicates a particle size of 5.93 ± 0.09 nm. S-6

7 Figure S4. Influence of ph of the buffer solution on the observed catalytic current spikes from single enzyme molecule collisions. Amperometric i-t curves in the presence of 0.1 U ml -1 enzyme solution in oxygen containing solution of 0.1 M acetate buffer, ph 4.0 (red curve) and ph 5.0 (black curve) and 0.05 M phosphate buffer, ph 6.0 (green curve) and ph 7.4 (blue curve), T=20 C. The AuUME was biased at V vs NHE. S-7

8 Supporting Note 2. Calculating turnover number from collision experiments. For turnover number calculations the impact spikes were analyzed using the Spike analyzer feature of the Origin v. 8.0 software ( for spike identification, height calculation and integration. Spikes were automatically identified at a threshold of 10 % of the highest spike from the user defined baseline, peaks height and area were calculated (Figure S5). The turnover number was calculated in two different ways: by using peak height and peak area. The peak height is related to the maximum speed of the reaction according to the following equation: i peak = Q t = Ne t (2) where i peak is the peak height, Q is the charge passing during time t, N is the number of electrons transferred in the reaction, e is the elementary charge. Considering that for the conversion of one substrate (oxygen) molecule four electrons are required, the maximum turnover number (k cat ) can be calculated as follows: k cat = N 4t = i 4e (3) The peak area was also used for an alternative calculation of the turnover rates using a similar approach. Dividing the obtained value of the spike area by the time width of the spike and the elementary charge gives the number of electrons transferred from the electrode to the enzyme molecule per second. The turnover number can be calculated by dividing the obtained value by 4. Figure S5C shows the obtained turnover rates distribution using the peak area for the calculations. We suggest that the signals observed were in the form of spikes rather than steady-state current steps due to partial denaturation or structural changes of the enzyme molecule as a result of the adsorption process. 6 Thus, calculation of peak current rather than peak area should give more relevant information about the turnover rates. Therefore, we used values obtained from the peak heights for all our further calculations and conclusions. S-8

9 Figure S5. Example of turnover number estimation from a collision experiment using the spike analyser in Origin. a. The figure shows an amperometric i-t curve (black) in the presence of 0.1 U ml -1 enzyme solution in an oxygen containing solution. Experimental conditions were: ph 5.0 (0.1 M acetate buffer), T=20 C. The AuUME was biased at V vs NHE. The red dashed line is a background used for spike identification, peak height calculation and integration. Identified spikes are marked with red markers. b. The table shows results of the automated peak identification and calculation. c. Distribution of turnover rates calculated from the collision experiments using the peak area fitted by log-normal statistics. The mean value is (1.02 ± 0.06) 10 5 s -1. S-9

10 Supporting Note 3. Dependence of electron transfer constant on potential. The electron transfer process from the electrode to the enzyme molecule can be written as follows: E ox + e k f E red (9) where E ox and E red are the oxidized and reduced forms of the enzyme respectively and k f is the rate of electron transfer at a certain applied potential. k f can be calculated from the Butler-Volmer equation for a reduction reaction: 10 k f = k 0 exp { αnf RT (E E 0)} (10) where k 0 is the standard heterogeneous rate constant, α is the transfer coefficient, E is the applied potential, E 0 is the standard potential of redox reaction, and (E-E 0 ) is the overpotential. The electron transfer coefficient α was assumed to be 1 for the irreversible enzymatic reaction (5), 10 n = 4, 11 and k 0 is estimated to be 9 s -1 for fungal laccase. 12 The k f at zero overpotential will be 9 s -1, which corresponds to a catalytic current, of only A. However, by increasing the overpotential just to 0.1 V, the k f will be ~5 10 7, which is larger than the intramolecular electron transfer rate. S-10

11 Supporting Note 4. Calculation of activation energy and reorganisation energy. The semi-classical expression for electron hopping described by Marcus theory is: 7 k et = 4π2 H h 2 λrt AB 2 exp { (ΔG0 +λ) 2 } (4) 4λRT where k et is a rate constant for electron transfer, h is the Planck s constant, R is the gas constant, T is the absolute temperature, λ is the reorganization energy required for the electron transfer, H AB is the electronic coupling matrix element between the donor and the acceptor, and ΔG 0 total Gibbs free energy change for the electron transfer reaction. A simplified form of eq. 4 can be written as: k et = k 0 exp { ΔG RT } (5) or for a one molecule as: k et = k 0 exp { ΔG kt } (6) is the where k is the Boltzmann constant and the activationless electron transfer rate constant, k 0, is described by: k 0 = exp{ β(r r 0 )} (7) where β = 1 Å -1 is the distance dependence of the decay of H AB, 8 r = 12 Å is the distance between the donor and the acceptor, 5 r 0 = 2.8 Å is the minimum distance between donor and acceptor. k 0 was calculated to be s -1 and ΔG was calculated to be 0.20 ev. ΔG is the activation energy for electron transfer: ΔG = (ΔG0 +λ) 2 4λ (8) The activation energy and the reorganization energy were calculated using eqs. 5-8 and ΔG 0 = 43 mev using a procedure described in the literature. 9 S-11

12 Figure S6. Example of a microelectrode surface check. Cyclic voltammogram of the AuUME in 1 mm 1,1'-ferrocenedimethanol in 0.1 M KCl, scan rate 50 mv s -1. S-12

13 References (1) Johannes, C.; Majcherczyk, A. Journal of Biotechnology 2000, 78, (2) Xiao, X.; Fan, F.-R. F.; Zhou, J.; Bard, A. J. Journal of the American Chemical Society 2008, 130, (3) (4) Benson, B. B.; Krause, D., Jr. Limnology and Oceanography 1980, 25, (5) Piontek, K.; Antorini, M.; Choinowski, T. Journal of Biological Chemistry 2002, 277, (6) Murgida, D. H.; Hildebrandt, P. Physical Chemistry Chemical Physics 2005, 7, (7) Marcus, R. A.; Sutin, N. Biochimica Et Biophysica Acta 1985, 811, (8) Gray, H. B.; Winkler, J. R. Quarterly Reviews of Biophysics 2003, 36, (9) Gupta, A.; Aartsma, T. J.; Canters, G. W. Journal of the American Chemical Society 2014, 136, (10) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications; Wiley, (11) Farver, O.; Wherland, S.; Koroleva, O.; Loginov, D. S.; Pecht, I. Febs Journal 2011, 278, (12) Jensen, U. B.; Vagin, M.; Koroleva, O.; Sutherland, D. S.; Besenbacher, F.; Ferapontova, E. E. Journal of Electroanalytical Chemistry 2012, 667, S-13