Biofilm sectional images and average RR spectra collected from electroactive biofilm at day 57 since inoculation under nonturnover conditions, with the electrode poised at 0(A, B, C, and D) or at −0.6 V (E, F, G, and H).

<p>Sectional maps (A, B, E, and F) are generated by binning the whole spectral information at 750 cm⁻¹ (A and E), and at 1641 cm⁻¹ (B and F), used here as markers of reduced and oxidized cytochromes, respectively. Each spectrum was averaged within the areas indicated by the dotted lines, representative of three biofilm locations, that is, top, middle, and bottom, respectively at a distance of 65, 40, and 15 µm from the electrode surface (not visible at the bottom of the spectral maps). The biofilm was exposed to the respective potentials for a period of at least 20 minutes to allow complete oxidation or reduction of cyt <i>c</i>, as suggested by stabilization of the current profile to very low levels (profiles shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089918#pone.0089918.s003" target="_blank">Figure S2A and B</a>).</p

Cyclic voltammograms (CVs) obtained from the electroactive biofilms.

<p>A) Nonturnover CVs recorded in acetate-depleted medium at scan rates ranging from 0.5 to 200 mV s⁻¹ (as indicated). B) Nonturnover CV recorded at the scan rate of 0.5 mV s⁻¹. The two redox couples <i>E_f,1</i> and <i>E_f,2</i> are centered at the formal potentials of −407 mV and −331 mV, respectively. <i>E_mean</i>, centered at −369 mV, indicates the arithmetic average of <i>E_f,1</i> and <i>E_f,2</i>. C) CVs of an electroactive biofilm metabolizing acetate (<i>i.e.</i>, turnover conditions), recorded at a scan rate of 0.5 mV s⁻¹. D) First derivatives of the turnover voltammetric curve performed at 0.5 mV s⁻¹. <i>E_f</i> represents the putative electron-transfer site, centered at −368 mV.</p

Raman sectional images and RR spectra averaged at different biofilm locations (as indicated by the dashed lines in the sectional maps) obtained from electroactive biofilms at different developing stages, grown at the electrode potential of 0 V vs. Ag/AgCl in the presence of 20 mM sodium acetate.

<p>Panels A, B, and C refer to a 10 days old biofilm with thickness <20 µm. Panels D, E, F, and G refer to an 80 days old biofilm with thickness >100 µm. Sectional maps are generated by binning over whole spectral information between 600 and 1800 cm⁻¹. Note that only the lower section of the 80 days old biofilm is shown in panel G. The biofilm thickness was in fact too large to be measured in its entirety in one single confocal measurement.</p

Typical Resonance Raman spectra of electroactive biofilm in the marker region between 550 and 1800⁻¹ obtained under different electrode polarizations in the absence of metabolic electron donor.

<p>A) RR spectra obtained with the working electrode poised at −0.6 V vs. Ag/AgCl. B) RR spectra obtained with the working electrode poised at 0 V vs. Ag/AgCl. C) Detail on RR spectra in the region 1250–1700 cm⁻¹. For clarity, the intensity of the spectra acquired at 0 V is multiplied by 2.</p

Example of combined voltammetry and RR measurements performed on a mature biofilm in acetate-depleted medium.

<p>RR spectra were collected continuously from a fixed portion of the biofilm positioned at approximately 50 µm from the electrode surface. A) current/potential vs. time (scan rate: 100 mV s⁻¹). B) time-series of the intensity of the RR spectra binned for the marker mode <i>ν</i>₁₅ at 750 cm⁻¹, and C) the marker mode <i>ν</i>₁₀ at 1640 cm⁻¹. D) RR spectra collected at time point 1 (0 s). E) RR spectra collected at time point 2 (17 s). F) RR spectra collected at time point 3 (28 s).</p

Biofilm sectional images and average RR spectra collected from electroactive biofilm at day 57 since inoculation, obtained after addition of 20 mM sodium acetate with the electrode poised at 0 V.

<p>Sectional maps are generated by binning the whole spectral information at (A) 750 cm⁻¹, and at (B) 1640 cm⁻¹, used here as markers of reduced and oxidized cyt <i>c</i>, respectively. Each spectrum was averaged within the areas indicated by the dotted lines, representative of three biofilm locations, that is, top, middle, and bottom, respectively at a distance of 65, 40, and 15 µm from the electrode surface (not visible at the bottom of the spectral sectional maps). Prior recording the RR spectra, the biofilm was exposed to the potential of 0 V vs. Ag/AgCl for 40 minutes to allow stabilization of the current profile (profiles shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089918#pone.0089918.s003" target="_blank">Figure S2C</a>).</p

Effect of the Protonation Degree of a Self-Assembled Monolayer on the Immobilization Dynamics of a [NiFe] Hydrogenase

Understanding the interaction and immobilization of [NiFe] hydrogenases on functionalized surfaces is important in the field of biotechnology and, in particular, for the development of biofuel cells. In this study, we investigated the adsorption behavior of the standard [NiFe] hydrogenase of Desulfovibrio gigas on amino-terminated alkanethiol self-assembled monolayers (SAMs) with different levels of protonation. Classical all-atom molecular dynamics (MD) simulations revealed a strong correlation between the adsorption behavior and the level of ionization of the chemically modified electrode surface. While the hydrogenase undergoes a weak but stable initial adsorption process on SAMs with a low degree of protonation, a stronger immobilization is observable on highly ionized SAMs, affecting protein reorientation and conformation. These results were validated by complementary surface-enhanced infrared absorption (SEIRA) measurements on the comparable [NiFe] standard hydrogenases from Desulfovibrio vulgaris Miyazaki F and allowed in this way for a detailed insight into the adsorption mechanism at the atomic level

Cytochrome <i>c</i> Provides an Electron-Funneling Antenna for Efficient Photocurrent Generation in a Reaction Center Biophotocathode

The high quantum efficiency of photosynthetic reaction centers (RCs) makes them attractive for bioelectronic and biophotovoltaic applications. However, much of the native RC efficiency is lost in communication between surface-bound RCs and electrode materials. The state-of-the-art biophotoelectrodes utilizing cytochrome <i>c</i> (cyt <i>c</i>) as a biological wiring agent have at best approached 32% retained RC quantum efficiency. However, bottlenecks in cyt <i>c</i>-mediated electron transfer have not yet been fully elucidated. In this work, protein film voltammetry in conjunction with photoelectrochemistry is used to show that cyt <i>c</i> acts as an electron-funneling antennae that shuttle electrons from a functionalized rough silver electrode to surface-immobilized RCs. The arrangement of the two proteins on the electrode surface is characterized, revealing that RCs attached directly to the electrode via hydrophobic interactions and that a film of six cyt <i>c</i> per RC electrostatically bound to the electrode. We show that the additional electrical connectivity within a film of cyt <i>c</i> improves the high turnover demands of surface-bound RCs. This results in larger photocurrent onset potentials, positively shifted half-wave reduction potentials, and higher photocurrent densities reaching 100 μA cm^–2. These findings are fundamental for the optimization of bioelectronics that utilize the ubiquitous cyt <i>c</i> redox proteins as biological wires to exploit electrode-bound enzymes

Substrate–Protein Interactions of Type II NADH:Quinone Oxidoreductase from <i>Escherichia coli</i>

Type II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins involved in respiratory chains and responsible for the maintenance of NADH/NAD⁺ balance in cells. NDH-2s are the only enzymes with NADH dehydrogenase activity present in the respiratory chain of many pathogens, and thus, they were proposed as suitable targets for antimicrobial therapies. In addition, NDH-2s were also considered key players for the treatment of complex I-related neurodegenerative disorders. In this work, we explored substrate–protein interaction in NDH-2 from <i>Escherichia coli</i> (<i>Ec</i>NDH-2) combining surface-enhanced infrared absorption spectroscopic studies with electrochemical experiments, fluorescence spectroscopy assays, and quantum chemical calculations. Because of the specific stabilization of substrate complexes of <i>Ec</i>NDH-2 immobilized on electrodes, it was possible to demonstrate the presence of two distinct substrate binding sites for NADH and the quinone and to identify a bound semiprotonated quinol as a catalytic intermediate

Orientation-Controlled Electrocatalytic Efficiency of an Adsorbed Oxygen-Tolerant Hydrogenase

<div><p>Protein immobilization on electrodes is a key concept in exploiting enzymatic processes for bioelectronic devices. For optimum performance, an in-depth understanding of the enzyme-surface interactions is required. Here, we introduce an integral approach of experimental and theoretical methods that provides detailed insights into the adsorption of an oxygen-tolerant [NiFe] hydrogenase on a biocompatible gold electrode. Using atomic force microscopy, ellipsometry, surface-enhanced IR spectroscopy, and protein film voltammetry, we explore enzyme coverage, integrity, and activity, thereby probing both structure and catalytic H₂ conversion of the enzyme. Electrocatalytic efficiencies can be correlated with the mode of protein adsorption on the electrode as estimated theoretically by molecular dynamics simulations. Our results reveal that pre-activation at low potentials results in increased current densities, which can be rationalized in terms of a potential-induced re-orientation of the immobilized enzyme.</p></div