Diego Millo

In Situ Spectroelectrochemical Investigation of Electrocatalytic Microbial Biofilms by Surface‐Enhanced Resonance Raman Spectroscopy

Metal-reducing bacteria not only play a key role in geochemical redox cycles, but also attract increasing attention in view of their relevance for microbial bioelectrochemical systems, a seminal sustainable technology. This growing research interest is triggered by the bacteria s capability to oxidize substrates such as acetate and to transfer the released electrons to an insoluble terminal electron acceptor, for example, iron-containing minerals in nature or a fuel cell anode in bioelectrochemical applications. The underlying electron-transfer (ET) mechanisms between the bacteria and the terminal electron acceptor may occur by different mechanisms, including direct and mediated electron transfer denoted as DET and MET, respectively. In the case of DET, the electrons are transferred from the respiratory chain in the cell to extracellular inorganic material via a complex architecture involving several outer membrane cytochromes (OMCs). These cytochromes are multiheme proteins whose function and number of heme groups may vary largely within the same family. Although several studies investigated the behavior of these proteins embedded in microbial biofilms of wild-type and mutant Geobacter sulfurreducens, the archetype bacteria family employing DET, the role of these cytochromes in the heterogeneous ET across the biofilm/electrode interface is far from clearly understood. This is particularly true since structural data are currently only known for two OMCs, namely, OmcF and OmcZ. 11] In this respect, spectroscopic techniques that can be applied to biofilms in situ may provide important structural information about the OMCs involved in the DET. To date, only two spectroscopic studies were devoted to the investigation of OMCs embedded in the cellular membrane. 13] The spectroscopic measurements of these works were carried out with washed and re-suspended cells, but did not refer to intact biofilms grown on an electrode. Herein, we present for the first time in situ spectroscopic characterization of OMCs in a catalytically active microbial biofilm. By measuring the electrochemical and spectroscopic properties of microbial cells embedded in their natural biofilm habitat, a more realistic picture on the natural electron transfer will be provided. Therefore, we have employed surface-enhanced resonance Raman (SERR) spectroscopy in combination with cyclic voltammetry (CV). SERR spectroscopy exploits the combination of the molecular resonance Raman (RR) and the surface-enhanced Raman (SER) effect to probe selectively the heme groups solely of the proteins in proximity of the electrode surface. 14] This powerful technique, in our case performed under strict electrochemical control, reveals the redox, coordination and spin states of the heme iron as well as the nature of its axial ligand, thereby providing important structural information that may complement the interpretation of electrochemical data obtained by CV. 16] The biofilms were grown at a constant potential on roughened (i.e. SER-active) silver electrodes using 10 mm acetate as substrate (see the Supporting Information for experimental details). These biofilms produced a maximum chronoamperometric current density of 600 mAcm 2 (Figure SI2 in the Supporting Information), which is in good agreement with previous studies using graphite anodes. The voltammetric behavior of the biofilms was monitored under turnover (Figure SI3) and nonturnover conditions [that is, with and without the substrate (e.g. acetate), respectively]. Figure 1 shows the CV behavior of such a biofilm for nonturnover conditions. The two redox couples that are proposed to be involved in the DET, Ef,1 and Ef,2, are centered at formal potentials of 282 mV and 363 mV, respectively (all potentials are reported versus the Ag/AgCl (3.0m KCl) reference electrode). The main overall shape and peak positions of the cyclic voltammogram shown in Figure 1 are very similar to those obtained on graphite electrodes in parallel experiments and in previous studies, showing that biofilm formation is not affected by the nature of the electrode material. The similarity between these CV traces and those obtained solely from biofilms of Geobacter sulfurreducens indicates that the biofilm is highly dominated [*] Dr. D. Millo, H. K. Ly, Prof. Dr. P. Hildebrandt Institut f r Chemie, Sekr. PC14, Technische Universit t Berlin Strasse des 17. Juni 135, 10623 Berlin (Germany) Fax: (+ 49)30-3142-1122 E-mail: diego.millo@tu-berlin.de

Surface-enhanced vibrational spectroscopy for probing transient interactions of proteins with biomimetic interfaces: electric field effects on structure, dynamics and function of cytochrome c

Most of the biochemical and biophysical processes of proteins take place at membranes, and are thus under the influence of strong local electric fields, which are likely to affect the structure as well as the reaction mechanism and dynamics. To analyse such electric field effects, biomimetic interfaces may be employed that consist of membrane models deposited on nanostructured metal electrodes. For such devices, surface‐enhanced resonance Raman and IR absorption spectroscopy are powerful techniques to disentangle the complex interfacial processes of proteins in terms of rotational diffusion, electron transfer, and protein and cofactor structural changes. The present article reviews the results obtained for the haem protein cytochrome c, which is widely used as a model protein for studying the various reaction steps of interfacial redox processes in general. In addition, it is shown that electric field effects may be functional for the natural redox processes of cytochrome c in the respiratory chain, as well as for the switch from the redox to the peroxidase function, one of the key events preceding apoptosis.

Combined Electrochemistry and Surface‐Enhanced Infrared Absorption Spectroscopy of Gramicidin A Incorporated into Tethered Bilayer Lipid Membranes

Support from the support: Tethered bilayer lipid membranes containing the cation-channel-forming peptide gramicidin A were assembled on nanostructured Au films. The combination of surface-enhanced infrared absorption (SEIRA) and electrochemical impedance spectroscopy (EIS) was used for the in situ structural and functional characterization of gramicidin A in the same device. Copyright

Role of the HoxZ subunit in the electron transfer pathway of the membrane-bound [NiFe]-hydrogenase from Ralstonia eutropha immobilized on electrodes.

The role of the diheme cytochrome b (HoxZ) subunit in the electron transfer pathway of the membrane-bound [NiFe]-hydrogenase (MBH) heterotrimer from Ralstonia eutropha H16 has been investigated. The MBH in its native heterotrimeric state was immobilized on electrodes and subjected to spectroscopic and electrochemical analysis. Surface enhanced resonance Raman spectroscopy was used to monitor the redox and coordination state of the HoxZ heme cofactors while concomitant protein film voltammetric measurements gave insights into the catalytic response of the enzyme on the electrode. The entire MBH heterotrimer as well as its isolated HoxZ subunit were immobilized on silver electrodes coated with self-assembled monolayers of ω-functionalized alkylthiols, displaying the preservation of the native heme pocket structure and an electrical communication between HoxZ and the electrode. For the immobilized MBH heterotrimer, catalytic reduction of the HoxZ heme cofactors was observed upon H(2) addition. The catalytic currents of MBH with and without the HoxZ subunit were measured and compared with the heterogeneous electron transfer rates of the isolated HoxZ. On the basis of the spectroscopic and electrochemical results, we conclude that the HoxZ subunit under these artificial conditions is not primarily involved in the electron transfer to the electrode but plays a crucial role in stabilizing the enzyme on the electrode.

Real-time measurements of the redox states of c-type cytochromes in electroactive biofilms: a confocal resonance Raman microscopy study

Confocal Resonance Raman Microscopy (CRRM) was used to probe variations of redox state of c-type cytochromes embedded in living mixed-culture electroactive biofilms exposed to different electrode polarizations, under potentiostatic and potentiodynamic conditions. In the absence of the metabolic substrate acetate, the redox state of cytochromes followed the application of reducing and oxidizing electrode potentials. Real-time monitoring of the redox state of cytochromes during cyclic voltammetry (CV) in a potential window where cytochromes reduction occurs, evidenced a measurable time delay between the oxidation of redox cofactors probed by CV at the electrode interface, and oxidation of distal cytochromes probed by CRRM. This delay was used to tentatively estimate the diffusivity of electrons through the biofilm. In the presence of acetate, the resonance Raman spectra of young (10 days, j = 208±49 µA cm−2) and mature (57 days, j = 267±73 µA cm−2) biofilms show that cytochromes remained oxidized homogeneously even at layers as far as 70 µm from the electrode, implying the existence of slow metabolic kinetics that do not result in the formation of a redox gradient inside the biofilm during anode respiration. However, old biofilms (80 days, j = 190±37 µA cm−2) with thickness above 100 µm were characterized by reduced catalytic activity compared to the previous developing stages. The cytochromes in these biofilm were mainly in the reduced redox state, showing that only aged mixed-culture biofilms accumulate electrons during anode respiration. These results differ substantially from recent observations in pure Geobacter sulfurreducens electroactive biofilms, in which accumulation of reduced cytochromes is already observed in thinner biofilms, thus suggesting different bottlenecks in current production for mixed-culture and G. sulfurreducens biofilms.

Analyzing the catalytic processes of immobilized redox enzymes by vibrational spectroscopies

Analyzing the structure and function of redox enzymes attached to electrodes is a central challenge in many fields of fundamental and applied life science. Electrochemical techniques such as cyclic voltammetry which are routinely used do not provide insight into the molecular structure and reaction mechanisms of the immobilized proteins. Surface‐enhanced infrared absorption (SEIRA) and surface‐enhanced resonance Raman (SERR) spectroscopy may fill this gap, if nanostructured Au or Ag are used as conductive support materials. In this account, we will first outline the principles of the methodology including a description of the most important strategies for biocompatible protein immobilization. Subsequently, we will critically review SERR and SEIRA spectroscopic approaches to characterize the protein and active site structure of the immobilized enzymes. Special emphasis is laid on the combination of surface‐enhanced vibrational spectroscopies with electrochemical methods to analyze equilibria and dynamics of the interfacial redox processes. Finally, we will assess the potential of SERR and SEIRA spectroscopy for in situ investigations on the basis of the first promising studies on human sulfite oxidase and hydrogenases under turnover conditions.

Unraveling the Interfacial Electron Transfer Dynamics of Electroactive Microbial Biofilms Using Surface‐Enhanced Raman Spectroscopy

The electron transfer (ET) processes of electroactive microbial biofilms have been investigated by combining electrochemistry and time-resolved surface-enhanced resonance Raman (TR-SERR) spectroscopy. This experimental approach provides selective information on the ET process across the biofilm-electrode interface by monitoring the redox-state changes of heme cofactors in outer membrane cytochromes (OMCs) that are in close vicinity (i.e., within 7 nm) to the Ag working electrode. The rate constant for heterogeneous ET of the surface-confined OMCs (sc-OMCs) of a mixed culture derived electroactive microbial biofilm has been determined to be 0.03 s(-1) . In contrast, according to kinetic simulations the ET between sc-OMCs and their redox partners, embedded within the biofilm, is a much faster process with an estimated rate constant greater than 1.2 s(-1) . The slow rate of heterogeneous ET and the lack of high-spin species in the SERR spectra rule out the direct attachment of the sc-OMCs to the electrode surface.

Vibrational stark effect of the electric-field reporter 4-mercaptobenzonitrile as a tool for investigating electrostatics at electrode/SAM/solution interfaces.

4-mercaptobenzonitrile (MBN) in self-assembled monolayers (SAMs) on Au and Ag electrodes was studied by surface enhanced infrared absorption and Raman spectroscopy, to correlate the nitrile stretching frequency with the local electric field exploiting the vibrational Stark effect (VSE). Using MBN SAMs in different metal/SAM interfaces, we sorted out the main factors controlling the nitrile stretching frequency, which comprise, in addition to external electric fields, the metal-MBN bond, the surface potential, and hydrogen bond interactions. On the basis of the linear relationships between the nitrile stretching and the electrode potential, an electrostatic description of the interfacial potential distribution is presented that allows for determining the electric field strengths on the SAM surface, as well as the effective potential of zero-charge of the SAM-coated metal. Comparing this latter quantity with calculated values derived from literature data, we note a very good agreement for Au/MBN but distinct deviations for Ag/MBN which may reflect either the approximations and simplifications of the model or the uncertainty in reported structural parameters for Ag/MBN. The present electrostatic model consistently explains the electric field strengths for MBN SAMs on Ag and Au as well as for thiophenol and mercaptohexanoic acid SAMs with MBN incorporated as a VSE reporter.

Spectroelectrochemical analyses of electroactive microbial biofilms

Understanding the mechanism of ET (electron transfer) through electroactive microbial biofilms is a challenge in the field of fundamental and applied life sciences. To date, electrochemical techniques such as CV (cyclic voltammetry) have been applied successfully to study the ET process in intact microbial biofilms on electrodes, providing important insight into their redox properties. However, CV as such does not provide any structural information about the species involved in the redox process. This shortcoming may limit the understanding of the ET process in microbial biofilms. To overcome this restriction, spectroelectrochemical techniques have been designed consisting of a spectroscopic technique performed in combination with electrochemical methods on the same electrode sample. These analytical approaches allow in vivo measurements of microbial biofilms under physiologically relevant conditions and controlled applied potential. The present review describes these spectroelectrochemical methodologies and critically addresses their impact on the understanding of the ET through biofilms.

Characterization of hybrid bilayer membranes on silver electrodes as biocompatible SERS substrates to study membrane-protein interactions

Hybrid bilayer lipid membranes (HBMs) were built on roughened silver electrodes exhibiting surface-enhanced Raman scattering (SERS) activity. The HBM consisted of a first layer of octadecanethiol (ODT) directly bound to the electrode surface, on which a second layer of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) was obtained by self-assembled phospholipid vesicle fusion. The electrochemical properties of the HBM were investigated in situ by cyclic voltammetry (CV), AC voltammetry and electrochemical impedance spectroscopy (EIS). The results indicate that our HBMs are well-formed, and their insulating properties are comparable to those observed for HBM supported by smooth metal substrates. The interaction between the bilayer and the human enzyme cytochrome P450 2D6 (CYP2D6) was investigated. Surface-enhanced resonance Raman scattering (SERRS) measurements in combination with AC and EIS, performed on the same electrode sample, proved that the CYP2D6 is immobilized on the HBM without evident alterations of its active site and without significant perturbations of the bilayer architecture. This study yields novel insights into the properties of HBMs built on roughened surfaces, providing in situ electrochemical characterization of a substrate which is suitable for studying peripheral membrane proteins with SERRS spectroscopy.