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Catalytic Activity and Proton Translocation of Reconstituted Respiratory Complex I Monitored by Surface-enhanced Infrared Absorption Spectroscopy Oscar Gutierrez-Sanz, Enrico Forbrig, Ana P Batista, Manuela M Pereira, Johannes Salewski, Maria Andrea Mroginski, Robert Goetz, Antonio L. De Lacey, Jacek Kozuch, and Ingo Zebger Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b04057 • Publication Date (Web): 19 Mar 2018 Downloaded from http://pubs.acs.org on March 20, 2018

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Catalytic Activity and Proton Translocation of Reconstituted Respiratory Complex I Monitored by Surface-enhanced Infrared Absorption Spectroscopy Oscar Gutiérrez-Sanz,#,‡ Enrico Forbrig,§,‡ Ana P. Batista, ⊥,  Manuela M. Pereira, ⊥,  Johannes Salewski,§ Maria A. Mroginski,§ Robert Götz,§ Antonio L. De Lacey,#* Jacek Kozuch,§,†* Ingo Zebger§* # §

Instituto de Catalisis y Petroleoquimica, CSIC c/ Marie Curie 2, 28049 Madrid, Spain

Technische Universität Berlin, Department of Chemistry, PC 14, Strasse des 17. Juni 135, D-10623 Berlin, Germany

⊥

Instituto de Tecnologia Química e Biológica – António Xavier, Universidade Nova de Lisboa, Av. da Republica EAN, 2780-157 Oeiras, Portugal

ABSTRACT: Respiratory Complex I (CpI) is a key player in the way organisms obtain energy, being an energy transducer, which couples NADH:quinone oxidoreduction with proton translocation by a mechanism that remains elusive so far. In this work, we monitored the function of CpI in a biomimetic supported lipid membrane (SLM) system assembled on a 4-aminothiophenol (4-ATP) self-assembled monolayer (SAM) by surface-enhanced infrared absorption spectroscopy (SEIRAS). 4-ATP serves not only as linker molecule to a nanostructured gold-surface but also as pH sensor, as indicated by concomitant density functional theory (DFT) calculations. In this way, we were able to monitor NADH:quinone oxidoreduction-induced transmembrane proton translocation via the protonation state of 4-ATP depending on the net orientation of CpI molecules induced by two complementary approaches. An associated change of the amide I/amide II band intensity ratio indicates conformational modifications upon catalysis which may involve movements of transmembrane helices or other secondary structural elements as suggested in literature [Di Luca et al., Proc. Natl. Acad. Sci. USA, 2017, 114, E6314-E6321].

Introduction Respiratory complex I (CpI, NADH:quinone oxidoreductase) plays a major role in the energy transduction processes, being part of the respiratory chain of mitochondria and many bacteria. It catalyzes the two electron oxidation of NADH and the reduction of quinone, coupled to charge translocation from the negatively charged side (N-side, prokaryotic cytoplasm or mitochondrial matrix) to the positively charged side (P-side, prokaryotic periplasm or mitochondrial intermembrane space) of the respective membrane.1 The charge translocation results in the formation of a transmembrane potential difference, fostering endergonic processes such as ATP synthesis, active transport or motility and is thus essential for life.1 The mitochondrial and bacterial CpI share 14 subunits, which compose the “minimal functional unit”. The mitochondrial complex also harbors several accessory subunits, summing up to 45 subunits.2 CpI contains two structural/functional domains; the hydrophilic peripheral arm, responsible for the catalytic activity and the membrane part in which ion translocation takes place.1,3–6 In bacteria, the latter has a curved shape with a total length of 180 Å and is constituted by seven hydrophobic subunits. The pe-

ripheral arm has a Y shape, a length of 130 Å and is composed of seven hydrophilic subunits.7 It also contains from 8 to 10 iron-sulphur clusters responsible for the electron transfer over nearly 100 Å from a flavin mononucleotide (FMN), which receives the electrons directly from NADH, to the quinone binding sites.7–9 Currently, one of the key questions about CpI function is how this coupling of electron transfer and proton translocation occurs, since the two processes take place in different parts of the enzyme, separated by large distances. This is suggested to be related to conformational changes involving shifts of helices at the quinone binding site at the interface of the peripheral and membrane domains and a movement of the peripheral arm.7,10–14 In this work, we aimed to monitor the process involved in this coupling mechanism by surfaceenhanced infrared absorption spectroscopy (SEIRAS). The reconstitution of purified membrane enzymes on surfaces modified with biomimetic membranes allows to study both, the catalytic process and associated structural rearrangements by a combination of methodologies.15 In our previous work, we functionally reconstituted the entire respiratory CpI from Rhodothermus marinus in a biomimetic supported lipid membrane system (SLM) on a

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4-aminothiophenol (4-ATP) self-assembled monolayer (SAM),16 while maintaining the enzyme’s activity with respect to both of its functions (NADH:quinone oxidoreduction and proton translocation). Here, we extended this investigation using the technique of SEIRAS to provide additional structural insights into the previously applied electrochemical approach. Although we made use of a more elaborate tethered bilayer lipid membrane system to investigate the generation of the transmembrane potential by cytochrome bo3 in a related study,17 here we have chosen this simpler setup due several reasons: (a) the possibility to obtain a much higher protein coverage, (b) less spatial restrictions of the aqueous reservoir that allows more efficient orientational control of CpI with its protruding arm and (c) the ability of 4-ATP to act as a spectroscopic pH sensor with much less spectral overlay than quinone/quinol difference bands in another approach, vide infra.17 SEIRAS is particular advantageous since structural and orientational changes in the protein and its surrounding can be readily monitored. The methodology has also been successfully applied in studies of several membrane proteins17–20 including CpI.21 However, in the study of Kriegel et al., CpI was immobilized with the hydrophilic domain facing the flat gold electrode,21 in an opposite orientation compared to our electrochemical reference study, by an engineered His-tag at a peripheral subunit or a NADH-SAM. SEIRAS is sensitive to IR absorptions of molecules in close vicinity to the nanostructured Au film. Similar to surfaceenhanced Raman, the main features of SEIRA can be explained based on the electromagnetic mechanism. Accordingly, the incident IR radiation excites collective electron resonances, referred to as localized surface plasmons, generating a polarized electromagnetic ﬁeld that leads to an enhancement of IR signals by 2-3 orders of magnitude. Since the enhancement decays with a distance dependence of d-6, it is insignificant at about 8 nm from the gold surface and contributions from the bulk solution can be neglected. In addition, this kind of polarized enhancement lays the basis for the surface-selection rule, that is a preferential enhancement IR modes with transition dipole moment changes perpendicular to the surface, while the corresponding vibrations, which are oriented parallel to surface do not contribute to the spectral intensity. For further details we refer to the work of Osawa.22 This fact can be used for an estimation of the orientation of ordered transmembrane helices of membrane proteins or enzymes. While the modes in the amide II region result from dipole moment changes along the membrane plane and perpendicular to helix axis, modes forming the amide I band are preferentially polarized along the helix axis.23,24 Thus, the ratio of amide I to amide II band intensity provides a quantity describing the orientation of proteins at SEIRA-active surfaces. If this ratio is higher than the isotropic/non-polarized ratio then a preferential orientation of helices perpendicular to the surface is

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expected, whereas in the opposite case helices are aligned preferentially along the surface.17,21,25–27

Experimental Enzyme purification, liposome and proteoliposome preparation Buffers and Rhodothermus marinus cell growth, CpI purification, liposome and proteoliposome formation for Cp I reconstitution in supported phospholipid bilayers were carried out as described previously.16,28,29 Surface Enhanced IR Absorption spectroscopic measurements SEIRA spectroscopic studies were performed using a trapezoidal-shaped ATR-IR element (W × L × H: 20 × 25 × 10 mm3). This silicon prism was treated as described by Miyake et al. to be covered by a nanostructured Au layer.30 After the electrochemical cleaning the geometric Au surface of 0.79 cm² was roughened by a factor of 2.1 leading to a surface area of 1.65 cm². In order to modify the gold surface, a Self-AssembledMonolayer (SAM) of 4-Aminothiophenol (Sigma) (4-ATP) was formed by overnight incubation in a 1 mM 4-ATP solution in ethanol. The liposome or proteoliposome suspensions were also deposited overnight on the 4-ATP-modified gold surface at 4 °C in presence of CALBIOSORB adsorbent Bio-Beads from Bio-Rad Laboratories, Inc. The measurements were performed in a Kretschmann-ATR configuration under an angle of incidence of 60°. All spectra were recorded in a spectral window of 4000 to 1000 cm-1 and with a resolution of 4 cm-1 using a Bruker Tensor 27 or a Bruker IFS66v/s spectrometer with a liquid nitrogen cooled photovoltaic or photoconductive MCT (Mercury Cadmium Telluride) detector, respectively. For each spectrum, 400 scans were accumulated. All chemicals were of highest purity grade available. Buffer solutions were prepared using Milli-Qwater with a resistance of > 18 MΩ cm. Vesicle preparation and (spectro-electrochemical) experiments were performed in buffer containing 20 mM Tris and 100 mM NaCl at pH 7.4. Proteoliposome preparation For the liposome preparation a volume of 200 μL of POPC in chloroform (1-palmitoyl-2-arachidonoyl-snglycero-3-phosphocholine, 10 mg/mL, Avanti Polar lipids) was mixed with 180 µL of POPA (1-palmitoyl-2oleoyl-sn-glycero-3-phosphate, 1 mg/mL, Avanti Polar Lipids) in chloroform and with 35 μL of 1.5 mg/mL 2,3-dimethyl-1,4-naphthoquinone (DMN) in a 50/50 chloroform/methanol (Sigma Aldrich) mix. This step was repeated three times followed by evaporation of the solution under a N2 stream and under vacuum overnight to removed traces of solvent. The addition of 100 mM PBS, pH 5.8 was added to form a 2 mg/mL suspension of phospholipids that was extruded with an extruder (Avanti Polar lipids) equipped with a porous membrane (100 nm pore diameter) leading to unilamellar liposomes.


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Two types of proteoliposomes were used: POPC:POPA (90:10) liposomes with and without DMN (50 µg/mL). In both cases, CpI was inserted in the same way: 10 μL a of 6.2 mg/mL CpI was added to 500 μL of the liposome suspension and stirred by vortexing, adding 15 mg of CALBIOSORB adsorbent biobeads (Calbiochem) every 45 min of stirring. The biobeads were added to remove the detergent from the complex I sample, thus facilitating its reconstitution in the liposomes. The biobeads were washed with 100 mM PBS, pH 5.5 before use. The freshly prepared unilamellar liposomes were added onto the 4-ATP SAM in 100mM PBS pH 7 buffer for 2-3 h to form the SLM respectively (1-step procedure). The addition of the proteoliposomes in the same way led to the construction of the SLM with incorporated CpI (2-step procedure).

Electrochemical measurements The SEIRA cell was also adapted to electrochemical studies, where the gold surface served as working electrode. An Ag/AgCl (3M KCl) electrode and a platinum mesh were used as reference and counter electrode, respectively. The measurements were performed on an Autolab PGSTAT12 potentiostat or a μ-Autolab II electrochemical analyzer controlled by GPES 4.9 software (Metrohm). Impedance spectra were measured using the FRA software in the frequency range of 0.05 Hz to 100 kHz at a DC potential of 0 mV and a rms-amplitude of 25 mV.

All experiments were reproducible.

DFT IR Spectra of 4-ATP To understand the two different pH regimes observed in the SEIRA difference spectra, various 4-ATP structures were calculated using density functional theory (DFT). Thereby, the thiol hydrogen was substituted by an Au atom to account for structural changes upon binding to the Au surface. Geometry optimization and vibrational analysis were performed on the BP86 level of theory31,32 using Gaussian 09.33 For C, H, N, and O atoms the 6-31g* basis set was chosen, for the heavier S the TZVP basis set, and for Au LanL2DZ (using a pseudo core potential) were employed.34,35 All geometry optimizations were performed using the keywords “opt=tight” and “int=ultrafine”. Geometry optimization using periodic boundary conditions (PBC) was performed by specifying two translation vectors that span the plane parallel to the supporting surface. As expected, imaginary frequencies (between 0 and -100 cm-1) resulted from the normal mode analysis based on this procedure, which were identified as torsional movements of whole 4-ATP molecules. This, however, is comprehensible and unavoidable because normal mode analysis cannot be performed together with PBC. Furthermore, the low

value (> -100 cm-1) of these frequencies shows that the frequency region of our interest (1400 – 1800 cm-1) can be reliably evaluated. Assignment of the calculated normal modes was obtained by evaluating the respective potential energy distributions.36,37 Only GaussView was used to generate the theoretical IR spectra from the calculation with a FWHM of 8 cm-1. All further treatments (e.g. plotting, calculating difference spectra, etc.) were performed using Origin 2016. No scaling factors were applied to the present calculations.

Results and Discussion Immobilization of CpI in the biomimetic membrane system on 4-ATP We used 4-aminothiophenol (4-ATP) as selfassembled monolayer (SAM) to cover a nanostructured SEIRA-active gold working electrode. Due to the protonable amino group and the related IR spectral features, 4-ATP serves as an excellent pH sensor to analyze pH changes at the SAM/membrane interface in combination with IR spectroscopy (see Figure 3, Figure SI 5 and Table SI 1).38 The formation of the 4-ATP SAM in ethanol was monitored by SEIRAS and showed 3 prominent bands at 1628 cm-1, 1595 cm-1 and 1488 cm-1 (Figure 1A) that can be ascribed to 4-ATP and its protonated form 4-ATP-H+ (see Figure SI 7, Figure SI 8 and Table SI 3). The broadened band at 1628 cm-1 is due to an overlap of δ(NH2) and δ(NH3) modes of both forms and the absorptions at 1595 cm-1 and 1488 cm-1 are dominated by a combination of aromatic ν(CC) and δ(CH) modes with minor contributions of the δ(NH2) mode from 4-ATP and mainly a mixture of δ(NH3) and δ(CH) of 4-ATP-H+, respectively. CpI embedded in the lipid bilayer was assembled using two different approaches, which led to complementary results. In the stepwise procedure, liposomes consisting of a mixture of ca. 8:1:1 POPC (1-palmitoyl-2-arachidonoyl-sn-glycero-3phosphocholine), POPA (1-palmitoyl-2-oleoyl-snglycero-3-phosphate) and DMN (2,3-dimethyl-1,4naphthoquinone) were deposited on top of the 4-ATP SAM to form a supported lipid membrane (SLM) system. In a second step, CpI was integrated into the bilayer fragments. The single step procedure used preformed proteoliposomes containing CpI and the PA-PC-DMN-mixture directly adsorbed onto the 4-ATP SAM. Figure 1 shows difference spectra from the stepwise assembly process of the biomimetic SLM system, where the corresponding SEIRA spectrum of the 4-ATP SAM in 100 mM phosphate buffered-saline (PBS) solution (pH 7) was chosen as reference. The SEIRA (difference) spectrum recorded after incubation with liposomes (Figure 1B) reveals weak spectral contributions of the lipids, namely the ν(CO) stretching vibration at 1732 cm-1 and the corresponding symmetric (s) and antisymmetric (as) aliphatic adsorption bands found at 2854, 2924, and 2958 cm-1 [νs(CH2), νas(CH2), and νas(CH3)], respectively.


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These band positions point to a high fraction of gauche conformers and thus to a high fluidity of the bilayer.39 The rather low band intensities can be explained based on the steep decay of the SEIRA effect within ca. 8 nm.22 When fragments of a bilayer membrane with a considerable amount of intact liposomes (size of about 100 nm) are formed on the SAM, only a small fraction of these lipids may directly interact with the SAM and in such way contribute to the SEIRA spectrum. The related difference bands at 1595 cm-1 and 1488 cm-1 are ascribed to the 4-ATP vibrations (vide supra) and appear due to an interaction with lipids. The negative band at ∼3500 cm-1 (ν(OH) stretching of water) occurs as consequence of a replacement of water molecules by lipid molecules.19,20 The corresponding weaker δ(OH) bending mode at ca. 1650 cm-1 overlaps with the δ(NH) of 4-ATP at about 1630 cm-1, leading to a negaFigure 1. Stepwise procedure: Difference spectra of (A) 90 min incubated Cpl in 0.1 M PBS minus bare SLM in 0.1 M PBS, at pH 7; (B) Addition of 0,3 µM NADH to CpI after buffer exchange in 0.1 M PBS minus SLM in 0.1 M PBS, at pH 7; (C) Addition of 0,3 µM NADH to CpI after buffer exchange in 0.1 M PBS minus CpI after buffer exchange in 0.1 M PBS, at pH 7; (D) a water spectrum on bare gold scaled to match the -1 ν(H2O) band at ca. 3500 cm ; (E) 4-ATP SAM in PBS 0.1 M exemplarily shown at pH 5 minus pH 7. Due to possible variations of the absolute SEIRA intensities between independent experiments we can only provide the qualitative information of the directionality of the net proton flow (see text).

Figure 2. SEIRA difference spectra of (A) 4-ATP SAM after 12 h of incubation in ethanol at 25 °C using bare gold in ethanol at 25 °C as reference; (B) Supported lipid membrane (SLM) in 0.1 M PBS minus 4-ATP SAM in 0.1 M PBS, at pH 5.8 (C) incubation of Cpl for 90 min in 0.1 M PBS minus SLM in 0.1 M PBS, at pH 7 (D) CpI after exchange with fresh 0.1 M PBS at pH 7. (E) Proteoliposomes harboring CpI after overnight incubation at 4 °C using 4-ATP as reference. Traces B to D were recorded at 25 °C. For a better comparison traces B and E were multiplied by a factor of 4 and 2, respectively.

tive band contribution around 1610 cm-1. Upon incorporation of CpI and the removal of the detergent by Bio-Beads, the liposomes are expected to disrupt and spread on the surface to form a more planar, but still at least partially fragmented SLM. This process is visualized as intensity increase of the symmetric and antisymmetric CH2/CH3 stretching vibrations at 2800-3000 cm-1and a decrease of the broad, negative water bands at ca. 3500 cm-1 and 1650 cm-1 (Figure 1C). At the same time, the protein backbone-related amide I and amide II bands at 1657 cm-1 and 1550 cm-1 increase,17 indicating the incorporation of CpI into the SLM system (Figure 1C), vide infra. The positions of the corresponding band maxima indicate a large spectral contribution of α-helices.40 The low relative intensity of ν(CO) at 1732 cm-1 (in comparison to the CH2/CH3 stretchings) can be rationalized based on the a unfavourable orientation for SEIRA spectroscopic detection, potentially induced by the presence of the CpI enyzme.20 The exchange of the supernatant solution induces a further intensity increase of the amide bands to ca. 2.5 and 2 mOD, respectively, while the band positions remain and a decrease of characteristic lipid bands is observed (Figure 1D).41–43 This can be rationalized on the basis of the removal of residual detergent traces, leading to a reorganization of CpI within the biomimetic SLM system with a higher content of protein at the SAM surface. The low intensity of lipid signals (ca. 0.6 mOD at 2924 cm-1)


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reflects the high protein density and indicates a coverage of ca. 80 % of a protein monolayer (based both on the amide I intensity of ca. 3 mOD obtained by Kriegel et al.21 for CpI and an intensity of ca. 4 mOD at 2926 cm-1 for other membrane systems17,20). It should be noted that, as shown by impedance spectra (Figure SI 4), the presence of the lipid layer in the chosen system does not provide efficient insulation in contrast to other biomimetic membrane systems with less defects.17,19,44 However, this assembly allows immobilizing proteins at a much higher density and utilizing the pH sensing ability of 4-ATP. The amide I band intensity in Figure 1D is larger than the corresponding amide II absorption, revealing a ratio of ca. 1.26. While a single helix oriented perpendicularly to the Au surface can yield a value of up to 16,19 in more complex membrane proteins harboring turns between the helices and further structural elements, which are immobilized in an oriented manner on an Au surface, this ratio lies in a range between ca. 1.2 (photosystem I24, cytochrome bo317 or the membranebound hydrogenase26,45) and 2.2 (sensory rhodopsin25). This observation is further supported by results obtained during orientation inversion of a highly oriented cytochrome c oxidase sample (immobilized via an N-terminal or C-terminal his-tag and reconstituted into a SLM system, where the detected ratio changes within this range. On this basis we can estimate that a ratio between ca. 1.2 and 0.8 (≈ 1/1.2) indicates a helical protein with diminished or random orientation, while for a ratio < 0.8 the helices are oriented preferentially in parallel to the surface (to our best knowledge, the latter has not been shown experimentally, yet). Note that these values are based on spectroscopic observations using SEIRAS, which differs from other polarized IR techniques due to the sharp distance (d) dependent surface enhancement scaling with a value of d-6 and thus, may provide shifted values. Consequently, we conclude that the here observed ratio of 1.26 reflects a preferentially perpendicular orientation of the transmembrane α-helices of CpI with respect to the surface and in such way a functionally relevant orientation of the transmembrane α-helices of CpI in the SLM system with the peripheral arm facing the bulk solution, see scheme 1A (vide infra). In the single step formation of the biomimetic SLM construct, the CpI containing PA-PCproteoliposomes were incubated overnight in the presence of Bio-Beads. Figure 1E depicts the SEIRA spectrum obtained after the deposition of the proteoliposomes using the spectrum of the 4-ATP SAM as a reference. The decrease of the absorbance in the region of ca. 3500 cm-1 is in agreement with the removal of adsorbed water molecules as discussed above. The positive bands at 1654 cm-1 and 1552 cm-1 can be assigned again to the amide I and amide II bands of CpI. The difference in positions of the amide bands

Figure 3. SEIRA spectroscopic pH-titration of the 4-ATP SAM in PBS 0.1 M in the range of pH 3 to pH 9 (shown here as black and red spectra) using the respective spectrum of pH 7 as reference. Phosphoric acid was used to acidify the bulk solution. The pH was measured in-situ with a pH electrode. The corresponding blue colored spectra are IR spectra calculated by a DFT approach (low vs. high pH) demonstrating the related spectral effects of the loss of vibrational coupling, or protonation of single 4-ATP molecules as described in the text and SI in more detail. The spectral region -1 < 1550 cm of the difference spectrum Δvibrational-coupling was magnified by a factor of 20.

of 2-3 cm-1 between the single step and stepwise procedure can, in principle, be ascribed to different orientations or arrangements of the protein on the electrode, which would in such way also result in SEIRA spectra of different CpI subunits. This is in line with the observation that no lipid-characteristic IR bands, such as ν(CH2/CH3) and ν(CO) stretchings at 28003000 cm-1 and ca. 1730 cm-1, are observed. The negligible intensity of the lipid bands leads to the conclusion that the bilayer is located in a larger distance relative to the surface, which could be explained by a distribution of the two possible orientations of CpI, i.e. with the peripheral arm facing either the bulk solution (scheme 1A) or the SAM (scheme 1C), respectively. In this scenario, the SEIRA spectrum would have a weaker contribution of the perpendicularly oriented transmembrane helices due to the surfacesensitive character of SEIRAS (detection limited to a distance of ca. 8 nm). Instead it would contain a major contribution of the secondary structure elements of the peripheral arm. This is in line with the observation of weaker amide I and II band intensities of 1.1 and 1.2 mOD, respectively, and in particular with the resulting altered amide I/II ratio of 0.93 lying in the range of random oriented helices.


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Scheme 1. Schematic representation of the biomimetic construct of the CpI-bilayer system adsorbed on a nanostructured gold electrode that is functionalized by a 4-aminothiophenol SAM (4-ATP). Depending on the orientation of CpI, the catalytic NADH oxidation-induced net proton translocation will yield in (A) acidification or (C) alkalisation of the space between bilayer membrane and SAM (by + + nH or -(n+1)H per NADH, respectively, with n considered to be 4, based on studies of the mitochondrial enzyme and 49 only related to protein contribution). Reoxidation of the quinone (B) is assumed to lead to a simultaneous acidifica+ tion by 1H per e . The ‘real’ structure of 4 ATP is described 46 by.

Monitoring the function of CpI in biomimetic membranes The successful incorporation of CpI in a functional relevant conformation within the biomimetic SLM system using the stepwise approach was demonstrated by measuring the electrocatalytic activity of the enzyme. Chronoamperometry was recorded at 200 mV (vs. 3 M KCl Ag/AgCl) and exhibited in average an increase of the oxidative catalytic current of ca. 0.4 µA after addition of NADH (0,3 µM) and proves that the protein readily exchanges electrons with the electrode via the DMN pool distributed within the bilayer (see Figure SI 1 left).16 SEIRA measurements support the observation of an active CpI and exhibit further information regarding (re)orientational as well as protonation changes of the SAM. Due to the large distance of the respective substrate binding sites to the metal surface, no direct spectral fingerprints of NADH or DMN binding could be detected. However, we observe a positive difference band at 1591 cm-1 and a negative difference band at 1489 cm-1 which can be assigned to reversible protonation of 4-ATP (Figure 2B and 2C and SI 1 right) due to the CpI-induced proton-translocation under turn-over conditions. Other spectral contributions from specific amino acids, such as aspartate or glutamate in this region40 can be excluded due to the larger distance of the enzyme to the surface compared to the SAM or bilayer. To verify the function of 4-ATP as a pH-dependent proton sensor, a pH-titration of 4-ATP from pH 9 to pH 3 was conducted and followed spectroscopically in the SEIRA difference mode (Figure 3). The spectrum recorded at pH 7 served as reference spectrum, in order to facilitate a comparison with the NADH-

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induced spectral changes of the CpI-bilayer system, which were also measured at a bulk pH 7. The difference spectra collected during the pH titration exhibit changes of the 4-ATP SAM related IR bands at ca. 1590 cm-1 and 1485 cm-1, and a broad difference band of the H2O related δ(OH)-bending vibration at ca. 1650 cm-1. In the basic pH regime from pH 9 to pH 7, a negative difference band at 1591 cm-1 and a positive one at 1487 cm-1 can be observed. At acidic pH values (pH 7 to 5) the spectral changes are reversed and a positive difference band at 1591 cm-1 accompanied by a negative difference band at 1487 cm-1 is found. Since the spectrum at pH 7 was used as reference these changes reflect a protonation induced process. In fact, as shown in Figure SI 5, the pH-dependent band intensities of the 1591 cm-1 absorption follow a broadened sigmoidal shape, revealing an inflection point at 6.4 ± 0.1, which is located in a similar range as the pKa determined by capacitance measurements (6.9 ± 0.5)47 and thus can be assigned to the pKa of the 4-ATP SAM. Surprisingly, at pH values lower than 5 the spectral features in the difference spectrum invert and a negative band at 1596 cm-1 as well as a positive band at 1484 cm-1 appear. To rationalize the observation of these two pH-dependent transitions of 4-ATP in the SEIRA spectrum, i.e. in the ranges of 5 ≤ pH < 9 and pH < 5, we performed DFT calculations to simulate the effect of protonation of 4-ATP on the IR spectrum. The blue spectrum labeled “ΔATP-prot.” in Figure 3 shows the most trivial case, that is the difference spectrum of protonation of 4-ATP (protonated 4-ATP vs. 4-ATP, see Figure SI 9). The signs of the main spectral features at 1611 cm-1(-) and 1481 cm-1(+) closely resemble the spectrum recorded at pH 3, so that we can assign the spectral features dominating at pH < 5 to the direct protonation of single 4-ATP molecules. To understand the spectral changes in the range of 5 ≤ pH < 9, we simulated several conceivable scenarios (e.g. protonation of pairs of 4-ATP molecules sharing the proton; protonation of H2O molecules bridging 4-ATP molecules; change of hydration of 4-ATP molecules etc.), which, however, failed to explain this regime and instead yielded results similar to the “ΔATP-prot.” spectrum in Figure 3. In a previous work on the electrochemical behavior of 4-ATP SAMs Raj et al.48 suggested the formation of cation radicals, i.e. [Au-SC6H4-NH2]+, at a potential of 0.44 V with very similar spectral features, as observed here between pH values of 9 and 5, that initiates a polymerization of the SAM. As shown in Table SI 3, in our pH titration the open circuit potential increases from 0.03 V to 0.23 V in this pH region, but does not reach potentials at which oxidation to the radical should occur (see peak X in Figure 1 in this reference). It is worth mentioning that the authors also demonstrate that with decreasing pH the oxidation potential drastically shifts towards more positive potentials making the oxidation process even less likely. Figure SI 11 shows the experimental SEIRA difference spectrum of the product of the oxidized 4-ATP SAM clearly demonstrating a spectral pattern unrelated to the spectra observed in the pH range of 5 - 9. Furthermore, Figure SI 12 depicts the simulated IR difference spectra associated with the pro-


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posed radical species displaying a much more complex pattern that is not in line with the recorded SEIRA spectra, so that we can rule out such species as a major contribution to the spectra. The only model capable of recapitulating the observed difference spectra using DFT calculation was the loss of vibrational coupling between individual 4-ATP molecules showing a matching spectral pattern with peaks at 1610 cm-1(+) and 1494 cm-1(-) (blue spectrum in Figure 3 labeled “Δvib.-coupl.”; see Figure SI 9). According to this scenario and assuming an entirely deprotonated 4-ATP SAM, the initial pH change leads to a strong perturbation of the structural homogeneity of the SAM and thus, to a severe loss of vibrational coupling (major contribution of Δvibrational-coupling). This process can be initiated by e.g. a few protonated 4-ATP molecules (minor contribution of ΔATP-protonation) or other pH-induced but spectroscopically undetectable species. Furthermore, the more molecules get protonated, the more dominant the spectral contribution of ΔATP-protonation becomes and superimposes the spectral effect of Δvibrational-coupling. This transition is apparent in the difference spectrum of pH 4 vs. pH 7, where both 4-ATP peaks exhibited first-derivative shaped difference bands. A comparison of the 4-ATP difference bands related to the NADH-induced spectral changes (1591 cm-1(+) / 1489 cm-1(-); Figure 2C) and those obtained in the 4-ATP titration experiment (1591 cm-1(+)/ 1487 cm-1(-) at moderate pH values of 5-9; 1596 cm-1(-)/ 1484 cm-1(+) at strong acidities at pH < 5) reveals a nearly perfect conformity of the spectral variations after NADH addition with those related to a protonation of the ATP SAM as displayed for example at pH values of 5 or 6 (Figure 2E; see Figure 3). This result is comprehensible, since it is rather unlikely that the proton translocation by CpI could induce a change by more than one to two pH units. Therefore, the NADH-induced SEIRA difference spectrum of the CpI-bilayer system (activity confirmed by chronoamperometry in Figure SI 1 left) with positive and negative intensities of the difference bands at 1591 cm-1 and 1489 cm-1, respectively, can be ascribed to a protonation of the 4-ATP SAM upon CpI activity. Due to possible variations of the absolute SEIRA intensities between independent experiments (and in particular Au film qualities), we cannot determine the pH shift quantitatively with satisfactory accuracy, and therefore only provide the qualitative information of the directionality of the net proton flow. It should be noted that due to the weak insulation of the membrane system, this pH change is perceivable only under turnover. The proton flux is related to two different sources, i.e. enzyme and quinone pool.17 The exact stoichiometry of transmembrane proton translocation by the bacterial CpI is not yet established, but is currently considered to be n = 4 per NADH molecule.49 Thus, one fraction originates from net proton translocation of CpI towards the SAM, which supports a preferential orientation of CpI within the biomimetic membrane

Figure 4. Difference spectra related to the reconstitution of SLM system with CpI within the single step procedure. (A) Addition of proteoliposome solution with 4-ATP SAM as reference; (B) Addition of 0,3 µM NADH to reconstituted SLM, with 4-ATP SAM as reference; (C) B minus A (D) 4-ATP at pH 8 minus 4-ATP at pH 7, multiplied by a factor of 2. Due to possible variations of the absolute SEIRA intensities between independent experiments we can only provide the qualitative information of the directionality of the net proton flow (see text).

system (+4H+ or +nH+ per NADH at the SAM/bilayer interface; scheme 1A), whereby the peripheral arm faces the bulk solution. This orientation is possible, since CpI was added to a preformed bilayer membrane/liposome system and thus should be incorporated into the biomimetic SLM system in such preferential manner. Neglecting the contributions of quinol reoxidation, a 50:50 mixture of both orientations of CpI within the bilayer membrane would have a basifying effect (-1 H+ per NADH; scheme 1) on the pH at the CpI-bilayer membrane/4-ATP SAM interface.17 Another fraction of the proton flux is induced by DMN. After reduction by the protein (and uptake of 2 protons), a certain amount of DMN will pass the electrons to the electrode to maintain its previous established redox equilibrium. This oxidation is associated with a maximum release of 2 protons per DMN molecule (DMNH2 → DMN + 2 e- + 2 H+; scheme 1B) at the bilayer membrane/SAM interface and will amplify the acidification. The ratio between both processes is dependent on the protein to DMN ratio (ca. 1:1.34), the ratio of both orientations of the protein, and the rate of DMNH2 oxidation at the electrode. In addition, we observe a simultaneous increase of the amide I and amide II band intensities combined with a change of the amide I/amide II ratio from 1.26 to 1.34 (Figure 2B). To exclude an overlap of the amide I band increase with a possible raise of O-H vibrational modes of water, a water spectrum is depicted in Figure 2D for comparison. The spectrum is scaled to the O-H-stretching region at


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3500 cm-1 of Figure 2C. It is obvious that the intensity increase of the corresponding O-H-bending mode at 1650 cm-1 is practically negligible. Thus, the broad positive band at ca. 1650 cm-1 in Figure 2B can only be related to an intensity increase in the amide I region. This rise may be an indicator for a reorientation of the α-helical and/or other secondary structural components of CpI as demonstrated in a recent theoretical work by Di Luca et al. where such structural changes were involved in a proton-channel formation.13 Besides conformational changes in the membrane part, charge-induced movements of or within the peripheral arm upon NADH and quinone binding were suggested, previously.11,12 However, for a direct study of involved possible conformational changes an in-depth analysis of a series of CpI mutants is required. The data obtained within the single step formation of the biomimetic membrane/CpI construct complements the previous interpretation obtained by the stepwise procedure. Upon addition of NADH a slight intensity decrease of the amide I band at 1654 cm-1 is detectable (Figure 4B) and the amide II band intensity at 1552 cm-1 remains constant. The resulting small change of the amide I/amide II ratio from 0.93 before NADH addition to 0.88 after NADH addition suggests that SEIRA probes the enzymes incorporated preferentially in a reverse orientation compared to the previous procedure (and since the change in ratio is less perceivable the changes occur at increased distance from the electrode) and indicates that a larger fraction of CpI interacts with the SAM via the peripheral arm (see scheme 1C).21 In analogy to the evaluation of the stepwise procedure we compared the SEIRA difference spectra after NADH addition with the spectral pH titration data of the 4-ATP SAM. Thereby, the corresponding SEIRA spectra in Figure 4A-C display upon addition of NADH difference bands at 1591 cm-1 (negative) and at 1487 cm-1 (positive), which change in a reversible manner. This result is in agreement with the related difference spectra of the pH titration at higher pH values (Figure 4D; see Figure 3) and indicates an alkalinisation of the SAM, related to a net proton flow towards the bulk solution. It further implies that defects in the biomimetic membrane system allow the diffusion of NADH to the interface, which is in line with a very small resistance of the biomimetic membrane system obtained from impedance spectra (Figure SI 4). This observation supports the previous interpretation that the observed intensity ratio and the shifted amide I and amide II band positions indicate a preferential “upsidedown” orientation of CpI (scheme 1C) within the reconstituted SLM construct. In this case, the net proton flux via the protein (-5H+ or -(n+1)H+ per NADH at the SAM/bilayer membrane interface for “upside-down” orientation; only protein contribution) exceeds the effect of re-oxidation of DMN at the electrode and thus, a major amount of CpI appears to be oriented with the peripheral arm towards the surface. In our previous work, this single step formation was employed on an Au(111) surface, thereby revealing an electrochemically detectable proto-

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nation of the 4-ATP SAM and thus, suggested a net orientation of active protein with the peripheral arm pointing to the bulk solution, as depicted in scheme 1A.16 Therefore, the observed difference in orientation can be attributed to the nanostructured surface of the SEIRA Au electrode that leads to less homogenously ordered biomimetic membrane system adsorbed on 4-ATP and residual intact vesicles.

Conclusions The complementary results of this work demonstrate that CpI incorporated into the biomimetic SLM system is catalytically active and translocates protons. It also shows the capability of the SEIRA (spectroelectrochemical) approach to monitor the two processes simultaneously. Depending on the type of construction procedure used for the biomimetic membrane system, i.e. the stepwise approach applying an incorporation of CpI into a bilayer membrane/liposome layer or the single step assembly using CpI-containing proteoliposomes, the preferential orientation of the protein was controlled while maintaining its electro-catalytic activity. Based on 4-ATP as a spectroscopic pH sensor, the NADH-induced catalytic transmembrane proton transfer leads to an acidification of the 4-ATP SAM when CpI was incorporated stepwise and thus oriented preferentially with the peripheral arm upwards. Adsorbing proteoliposomes directly onto the SAM provides a major CpI-fraction that is oriented upside-down resulting in an alkalinisation of the SAM under turn-over conditions. Importantly, the two constructs report changes of the amide I/II band intensity ratio, which may reflect structural changes of CpI upon catalysis and proton translocation. Based on recent literature reports7,10–14 both, transmembrane α-helices and the interfacial region to the peripheral arm are proposed to experience pronounced structural changes. Thus, this work will pave the way for future structural and functional studies on the activity of CpI reconstituted in bilayer lipid membranes.

ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org and contains electrochemical and SEIRA spectroscopic control experiments, a pH-titration of the 4-ATP SAM, the DFT IR spectra of 4-ATPas well as the corresponding optimized DFT geometries.

AUTHOR INFORMATION Corresponding Author * [email protected] * [email protected] * [email protected]

Author Contributions ‡These authors contributed equally. Present Addresses


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†Department of Chemistry, Stanford University, Stanford, California 94305-5012, United States Departamento de Química e Bioquímica, Faculdade de Ciencias, Universidade de Lisboa, 1740-016 Lisboa, Portugal  iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal

Notes The authors declare no competing financial interest.

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ACKNOWLEDGMENT The work was funded by Fundação para a Ciência e a Tecnologia (IF/01507/2015 to MMP) and by the Spanish MINECO/FEDER project CTQ2015-71290-R. The project was supported by LISBOA-01-0145-FEDER007660 co-funded by FEDER through COMPETE2020-POCI and by Fundação para a Ciência e a Tecnologia. EF, JS, MAM, JK, and IZ thank the Deutsche Forschungsgemeinschaft (DFG) for financial support by the Cluster of Excellence EXC 314 “UniCat”; IZ also acknowledges the priority program 1927 “Iron-sulfur for Life” (DFG). JK also expresses his thanks to the DFG (DFG Forschungsstipendium KO 5464/1-1). We kindly thank Marius Horch for helpful discussions. OG-S thanks the MINECO for BES-2010035416 grant.

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