Bioelectronic circuit on a 3D electrode architecture: Enzymatic

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Bioelectronic circuit on a 3D electrode architecture: Enzymatic catalysis interconnected with photosystem I Dmitri Ciornii, Marc Riedel, Kai Ralf Stieger, Sven Christian Feifel, Mahdi Hejazi, Heiko Lokstein, Athina Zouni, and Fred Lisdat J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b10161 • Publication Date (Web): 01 Nov 2017 Downloaded from http://pubs.acs.org on November 1, 2017

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Journal of the American Chemical Society

Bioelectronic circuit on a 3D electrode architecture: Enzymatic catalysis interconnected with photosystem I Dmitri Ciornii*,1, Marc Riedel1, Kai R. Stieger1, Sven C. Feifel1, Mahdi Hejazi2, Heiko Lokstein3, Athina Zouni2, Fred Lisdat*,1 1

Biosystems Technology, Institute of Applied Life Sciences, Technical University of Applied Sciences Wildau, Hochschulring 1, 15475 Wildau, Germany 2 Biophysics of Photosynthesis, Institute for Biology, Humboldt-University of Berlin, Philippstrasse 13, Haus 18, 10115 Berlin, Germany 3 Department of Chemical Physics and Optics, Charles University, Ke Karlovu 3, 121 16 Prague, Czech Republic Supporting Information are available under:………………… ABSTRACT: Artificial light-driven

signal chains are particularly important for the development of systems converting light into a current, into chemicals or for lightinduced sensing. Here, we report on the construction of an all-protein, light-triggered, catalytic circuit based on photosystem I, cytochrome c (cyt c) and human sulfite oxidase (hSOX). The defined assembly of all components using a modular design results in an artificial biohybrid electrode architecture, combining the photo-physical features of PSI with the biocatalytic properties of hSOX for advanced light-controlled bioelectronics. The working principle is based on a competitive switch between electron supply from the electrode or by enzymatic substrate conversion.

Integration of biomolecules into an electrical circuit in which electrons can be routed in a desirable way represents an interesting topic in biomolecular electronics. It opens new perspectives for practical applications, where desired reactions can be triggered on demand or supplied with the needed energy. Thus, the design of functional biohybrid architectures on the nanoscale has gained intense research 1-4 interest over the last decades. Such biohybrids are based on an efficient biomolecule-electrode contact. This can be achieved via free or bound redox compounds, shuttling 5,6 electrons between the electrode and the biocatalytic entity 7,8 or by direct electron transfer. Efficient wiring of several biocatalysts with the electrode and with each other, however, remains challenging. One possibility for communication between multiple enzymes has been exploited by using reaction intermediates and establishing enzyme cascades, enzyme competition or 9-11 Metabolic channeling recycling schemes on electrodes. can be considered as a further advancement by constructing multi-enzyme complexes in an artificial way with reduced 12,13 diffusion pathways. Another step in the development of multi-protein systems represents the establishment of direct electron exchange between immobilized proteins on 14-16 electrode surfaces. Here the capability of natural redox

proteins to communicate even with non-native partners can 17 be exploited for the design of artificial signal cascades. The successful integration of light-sensitive proteins with biocatalysts is a research target for which only recently first examples have been demonstrated, where biological lightconverting complexes such as photosystem I (PSI) have been 18,19 The photo-active incorporated in biohybrid systems. complex PSI has also been successfully coupled with a hydrogenase via a dithiol-linker allowing a photocatalytically 20 driven electron supply for the enzyme . Following this idea it has been recently shown that hydrogen production and photocurrent production are feasible by combining PSI with 21 a hydrogenase via a redox polymer on an electrode. In a different approach the enzyme glucose oxidase has been coupled to an electrode-fixed PSI resulting in enhancement 22 of the anodic photocurrent in the presence of glucose. These developments may illustrate that the combination of biocatalytic conversions with photoactive entities are advantageous in connecting complex redox reactions since light and electrode potential can be used to control the processes. Encouraged by previous studies on photoelectrodes we have developed a modular self-driven photobiocatalytic architecture, in which the photo-active unit, PSI, produces a light-induced current, human sulfite oxidase (hSOX) acts as an electron supplier for PSI and cytochrome c (cyt c) works as a molecular wire between the bio-compounds and also towards the electrode. In our system the assembly of several biomolecules results in an efficient interprotein electron transfer allowing the establishment of well-defined electron pathways. For the incorporation of the multi-protein system we have used 3D inverse opal ITO (IO-ITO) electrodes (Fig. S1, SI) applying the previously reported template-based preparation 23 procedure. IO-ITO electrodes provide a high surface area, which in turn allows for harboring large amounts of biomolecules. In this system protein binding has been ensured without the need for polymers or mediators, but solely by adsorption due to the hydrophilic surface of such IO-ITO structures. Here a sequential incubation procedure starting with PSI and hSOX followed by cyt c has been used (see SI for details). It has to be emphasized that the idea is

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not a protein immobilization on top of each other, but to use the small redox protein cyt c to fill the space between the two immobilized biocatalysts allowing to act as molecular wire in between the proteins and towards the electrode. In order to verify the successful integration of all components into the IO-ITO structure, firstly, cyclic voltammetry (CV) has been applied. As shown in Fig. 1, the IO-ITO•PSI•hSOX•cyt c electrode exhibits a quasi-reversible redox behavior of cyt c with a formal potential at around 0.02 V vs. Ag/AgCl. From these experiments a surface -2 coverage of 17 ± 3 pmol*cm cyt c can be calculated (related to the electrochemically active area). The results do not only demonstrate the presence of cyt c, but also the electroactivity of a large amount of this redox protein, representing one of the pre-conditions for the successful delivery or uptake of electrons for the catalytic units. Upon illumination of the electrode a cathodic photocatalytic current is detected, starting at the redox potential of cyt c (Fig. 1) This verifies that PSI can accept electrons from cyt c upon illumination – even when another protein is present within the macroporous electrode structure. In the dark, after 2addition of the substrate of hSOX, SO3 , an anodic bioelectrocatalytic current can be observed (Fig. 1). Here, electrons are collected by the enzyme and shuttled via cyt c to the electrode. It has to be mentioned here that IOITO•hSOX•cyt c electrodes, i.e. without PSI, do not show any photocurrent under illumination (see SI, Fig. S2). Moreover, IO-ITO•PSI•cyt c electrodes, i.e. without hSOX, display no 2anodic response in the presence of SO3 (see SI, Fig. S3). The quantification of immobilized PSI by chlorophyll 24 -2 extraction yields a PSI coverage of 0.2 ± 0.01 pmol*cm . The hSOX coverage has been estimated by eluting the enzyme from the 3D IO-ITO to be in the range of 1.4 ± 0.4 -2 pmol*cm (see SI). The surface concentration data of all three proteins implicate a good coverage of the inner surface with the biomolecules. For PSI and cyt c the coverage reaches values in the range of a monolayer.

Figure 1. Cyclic voltammetry of an IO-ITO•PSI•hSOX•cyt c electrode. Blue curve – cathodic photocatalysis upon illumination. Red curve – enzymatic catalysis upon addition of 1 mM of Na2SO3. Inset shows an SEM image of the prepared 3D electrode used for protein incorporation. (5 mM potassium phosphate buffer pH 7, 10 mV/s, white light 100 2 W/m , reference: Ag/AgCl, 1 M KCl).

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From the catalytic currents and the determined amount of immobilized enzymes additionally the turnover numbers (Tn) of PSI and hSOX within the architecture can be -1 -1 calculated: Tn(PSI) = 16 ± 3 s and Tn(hSOX) = 1.6 ± 0.2 s . The Tn for PSI is comparable to the value for PSI immobilized on ITO without hSOX and the Tn for hSOX is in the range of values reported in literature (for hSOX 0.85 s 1 25 -1 26 and for chicken SOX (cSOX) 2-4 s ). This indicates that both catalysts react rather undisturbed by the presence of the other biocatalyst. Besides the functional assessment of each biomolecule separately we have further performed photochronoamperometric experiments with both catalytic components active. Here, the activity of hSOX is “switched on” by addition of the substrate, whereas the activity of PSI is “switched on” by illumination. When the electrode is polarized at -0.15 V vs. Ag/AgCl a clear cathodic photocurrent is obtained which decreases in the presence of 2increasing concentrations of SO3 (Fig. 2, A). When we carry out the same experiment, but with an electrode lacking hSOX, only a slight decrease of the cathodic photocurrent can be detected, which starts at higher sulfite concentrations (Fig. 2, B). The suppression of the cathodic photocurrent upon addition of sulfite to the IO-ITO•PSI•hSOX•cyt c electrode can therefore not simply be explained by putative side reactions of sulfite with PSI, cyt c or the ITO electrode, but has to be driven by the enzymatic reaction of hSOX in the structure. Concluding, there are two competing signal cascades which can be expressed as follows: (1) Electrode reaction: Electrode → cyt c → (cyt c)n →PSI → O2 (2) Enzyme reaction: 2SO3 → hSOX → cyt c → (cyt c)n → PSI→ O2 As depicted in equations (1) and (2) oxidized cyt c can receive electrons either from the electrode or from hSOX. If 2SO3 is added, a competing electrical circuit is established, since now the oxidized cyt c is not only reduced by the electrode, but also by the enzyme (pathway 2). This leads to a suppression of the electrode pathway (1) and hence, to a diminished photocurrent. The degree of suppression of the cathodic photocurrent is determined by the efficiency of competition between both pathways and provides a quantitative feedback on the sulfite concentration in solution. By plotting the photocurrent decrease vs. the concentration of added substrate a Michaelis-Menten-type behavior can be observed (Fig. 2, C); curve fitting gives rise to an apparent KM value of 59 + 5 µM. This value is higher 27 than previously reported for the enzyme in solution (1 µM) but is comparable to values reported for hSOX, immobilized 25 28 on electrodes (60 µM, 72 ± 14 µM ). These experiments show that the suppression of the photocurrent is of enzymatic origin. The photocurrent suppression reaches about 70 % of the initial photocurrent illustrating the rather high efficiency of the three protein system since pathway 2 can overtake the electron supply by pathway 1 to a very large extent. This also means that the enzyme reaction can “feed” the photoreaction at PSI with electrons.


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Figure 3. Schematic illustration of electron pathways within the biohybrid architecture upon illumination and addition of sulfite. Yellow arrows represent the light-induced electron cascade initiated by the charge separation process within PSI. Blue arrows represent the electron pathway during the oxidation of sulfite. The reaction of human sulfite oxidase generates electrons, which reduce oxidized cyt c. A competitive situation is thus generated at cyt c (see red circle), the cathodic photocurrent is suppressed and the degree of competition is detected at the electrode. The scheme also points to a high cyt c concentration at the luminal side of PSI. (Crystal structures – see SI). It has to be mentioned here that this concept also includes the possibility of cyt c-cyt c self- exchange and thus, multiple molecules of the redox protein are involved in the electron delivery from the electrode to PSI and also from the enzyme 29,30 to PSI.

Figure 2. Photocurrent behavior after addition of different concentrations of sulfite for IO-ITO•PSI•hSOX•cyt c (A) and for IO-ITO•PSI•cyt c (B) electrodes. (C) Plot of the change of the photocurrent density vs. the concentration of sulfite for IO-ITO•PSI•hSOX•cyt c (red dots) and for IO-ITO•PSI•cyt c (black squares). (stirred solution, 5 mM potassium phosphate buffer pH 7, -0.15 V vs Ag/AgCl, 1 M KCl, white 2 light 100 W/m ). Moreover, the oxidation of sulfite, which leads to an anodic current at higher potentials (Fig. 1), can now be transferred to a cathodic current measurement taking place at negative electrode potentials.

With these functional bio-compounds in the macroporous electrode we are able to transfer their function into a bioelectronic circuit, whereby the different reactions can be attributed to the following electronic elements: the trigger (sulfite) , the detector (electrode), the relay network, which also works as a Nernstian capacitor (cyt c) and the photodiode (PSI). The principle including the reaction pathways is illustrated in Fig. 3. Untill now we have described the electron transfer reactions of this bioelectronic system. Nonetheless, the system should be able to operate without any external driving force. In order to test this self-driven modus, open circuit potential (OCP) measurements have been performed. As depicted in Fig. 4, illumination of the IOITO•PSI•hSOX•cyt c electrode in the absence of sulfite leads to a slight increase of potential since this is a result of a complete oxidation of cyt c molecules by excited PSI (the increase is small since at the start of the measurement a large portion of cyt c is already oxidized). By subsequent addition of sulfite in the dark the OCP drops rapidly by about 200 mV. Here, the enzyme reaction is “switched on” and subsequently cyt c is reduced.


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surrogate electron delivery to PSI without any external energy source. This tri-protein architecture within a 3D electrode matrix demonstrates that well-defined electron pathways can be generated on an artificial platform. The system works as a bio-circuit and may thus stimulate further developments of smart bioelectronics devices.

ASSOCIATED CONTENT Supporting Information Supporting information contains materials and calculation methods. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected], Tel.: +49(0) 3375508456/799, Fax: +49(0)3375508578, www.th-wildau.de/lisdat *E-mail: [email protected], Tel.: +49(0)3375508353

Notes The authors declare no competing financial interests. Figure 4. Potentiometric measurements of an IOITO•PSI•hSOX•cyt c electrode (blue line) and IOITO•PSI•cyt c electrode (black line). Yellow areas indicate illumination periods. (5 mM potassium phosphate buffer pH 7, 1 mM sulfite). After exchange to pure buffer the OCP is 2 measured again under illumination (100 W/m ) but without sulfite in solution. This behaviour is only found for hSOX containing electrodes, not for electrodes where hSOX is absent. Here, the OCP remains constant, again demonstrating that electron supply occurs via the sulfite reaction at the enzyme. If the light is “switched on” again, the OCP increases and stabilizes between the fully oxidized and reduced state of cyt c. This steady-state depends on the rate of photo-oxidation compared to the rate of enzymatic reduction. When the biocatalytic electron supply is stopped (by removal of sulfite) and the electrode is still illuminated, the OCP returns to its initial value, indicating the complete oxidation of cyt c by PSI. These biomolecular reactions can be monitored at the electrode, since the OCP is defined as the ratio of the redox states of cyt c (cyt cox / cyt cred). This is reasonable, since it is the only component which can rapidly exchange electrons with the electrode and is present in high concentration on the surface. The potential measurements confirm the selfdriven character of this artificial photobiocatalytic system. In conclusion, in the present study the design of a multifunctional photobiocatalytic architecture has been shown. This system displays four distinct features: (i) Due to different triggers, such as light, substrate and potential, different reaction pathways can be switched “on” and “off” on demand, (ii) Photo-switchable sensing of sulfite is feasible since the photocurrent follows the sulfite concentration, but can be detected as a cathodic signal at negative polarisation, (iii) Due to the different reactions in the dark and under illumination the multi-biomolecular unit can be used as a capacitor, which is charged by the biocatalytic reaction and discharged by the photocatalytic process and (iv) The system demonstrates self-driven character and can be used to

ACKNOWLEDGMENTS We greatfully acknowledge financial support by the BMBF, Germany (Biotechnology 2020+, projects: 031A154A+B). We also thankful to Prof. Silke Leimkühler for the supply of the enzyme human sulfite oxidase.

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Bioelectronic circuit on a 3D electrode architecture: Enzymatic

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