Functional Vibrational Spectroscopy of a Cytochrome <i>c</i> Monolayer: SEIDAS Probes the Interaction with Different Surface-Modified Electrodes

Electrochemically induced infrared difference spectra of cytochrome c on various chemically modified electrodes (CMEs) are recorded by exploiting the surface-enhancement exerted by a granular gold film. We have recently developed surface-enhanced infrared difference absorption spectroscopy (SEIDAS), which provides acute sensitivity to observe the minute enzymatic change of a protein on the level of a monolayer. By these means, we demonstrate that the relative band intensities in the potential-induced difference spectra of adsorbed cytochrome c are significantly dependent on the type of CME used (mercaptopropionic acid, mercaptoethanol, 4,4‘-dithiodipyridine, or l-cysteine). These differences are attributed to the altered interaction of cytochrome c with the headgroup of the various CMEs leading to variations in surface orientation and relative distance from the surface. Nevertheless, the peak positions of the observed bands are identical among the CMEs employed. This implies that the internal conformational changes induced by the redox reaction of the adsorbed cytochrome c are not disturbed by the interaction with the CME and that full functionality of the protein is retained. Finally, we critically discuss our results within the framework of the different models for cytochrome c adsorption on CMEs

Orientational Control of the Physiological Reaction of Cytochrome c Oxidase Tethered to a Gold Electrode

The physiological reaction of a membrane protein is reconstituted on a solid-supported electrode by orientational control via the position of an affinity tag. Recombinant cytochrome c oxidase (CcO) from Rhodobacter sphaeroides is immobilized on a chemically modified gold surface via the affinity of a histidine tag (His-tag) to a nickel chelating nitrilotriacetic acid surface. Control of the orientation is achieved by the adsorption of CcO through the His-tag engineered into the two opposite sites of the membrane protein surface. After reconstitution into a lipid layer, the functionality of this enzyme film electrode is probed by surface-enhanced infrared absorption spectroscopy and cyclic voltammetry. We demonstrate that cytochrome c (Cc) binds and initiates the catalytic reaction of CcO only when the latter is orientated with subunit II facing the bulk aqueous phase while Cc does not interact with the oppositely orientated CcO. We infer from the observed catalytic dioxygen reduction at potentials below 240 mV (vs a normal hydrogen electrode) that reduced Cc mediates electron input into CcO in a way similar to the physiological pathway. The quantitative analysis of the IR spectra indicates the presence of an inactive population of Cc bound to CcO at equal amounts as the redox-active population. This methodological approach demonstrates that the orientation of the membrane protein can be controlled depending on the position of the affinity tag. The approach is considered to be of general applicability as the introduction of affinity tags is routine in current biochemistry

Influence of the Molecular Structure of Carboxyl-Terminated Self-Assembled Monolayer on the Electron Transfer of Cytochrome c Adsorbed on an Au Electrode: In Situ Observation by Surface-Enhanced Infrared Absorption Spectroscopy

Surface-enhanced infrared adsorption spectroscopy (SEIRAS) was employed for the in situ observation of structural changes that occur in a self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (MUA) bound on a gold surface. The observed SEIRA spectra reveal a deprotonation of the carboxyl head group of the MUA-SAM layer after adsorption. An analysis of the vibrational spectra suggests that the deprotonation process occurs when the adsorbed MUA molecules reach a critical mutual distance. MUA-SAMs promote direct electron transfer between the metal electrode and cytochrome c, the electron mediator between the integral membrane protein complexes of the respiratory chain. The results show that the coverage of cytochrome c increases with the coverage of deprotonated MUA on the surface. On the other hand, the electron transfer of cytochrome c is optimized only when a moderate amount of the carboxyl head group is deprotonated. The electron transfer of cytochrome c is suppressed with a further increase of the deprotonated MUA. The relationship between the surface structure of the MUA layer and the electron transfer of cytochrome c is discussed on the basis of the spectroscopic data

Molecular Impact of the Membrane Potential on the Regulatory Mechanism of Proton Transfer in Sensory Rhodopsin II

Metabolism establishes a potential difference across the cell membrane of every living cell which drives and regulates secondary ion and solute transfer across membrane proteins. Unraveling the effect of the membrane potential on the level of single molecular groups of the membrane protein was long hampered by the lack of appropriate analytical techniques. We have developed Surface Enhanced Infrared Difference Absorption Spectroscopy (SEIDAS), a highly sensitive vibrational technique for surface analysis, for the study of solid-supported monolayers of orientated membrane proteins. Here, we present spectroscopic data on vibrational changes of sensory rhodopsin II from Natronomonas pharaonis (NpSR II). The application of the electrode potential provides a voltage drop across the NpSR II monolayer through the Helmholtz double layer that mimics the cellular membrane potential. IR difference spectra indicated a shift of the photostationary equilibrium from an M and O mixture toward an M dominant equilibrium. The shift of positive to negative potential exhibited similar effects on the light-induced SEIDA spectra as the increase in pH. This effect is explained in terms of local pH change raised by the compensation of excess charge from the electrode. As we have shown earlier (Jiang, et al. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (34), 12113−12117), the application of an electric field opposite to the physiological proton transfer from the retinal Schiff base to its counterion Asp75 leads to the selective halt of the latter. However, when the solution pH is much higher than 5.8, that is, when the proton releasing group at the extracellular side is ionized, proton transfer of Asp75 becomes insensitive to the electric field exerted by the electrode. We infer that the deprotonation of the proton release group creates a local polar environment surrounding Asp75 as a consequence of hydrogen-bonding rearrangements that exceeds the energy of the external dipole. Our results reveal a molecular model for the physiological regulation of the photocycle of NpSR II by the potential drop across the membrane which came about by the interplay between the change in local pH at the membrane surface and the external electric field

Schematic domain drawing of aureochrome 1 and YtvA.

<p>Domain organization of full-length aureochrome 1 from <i>Vaucheria frigida</i> and of YtvA from <i>Bacillus subtilis</i>. The LOV domains are coloured in blue, while the effector domains are colored in orange. The J_α-helix is located C-terminal to the LOV domains (shown as a black bar).</p

Sequence alignment of aureochrome 1 with GCN4.

<p>Sequence alignment of the bZIP domains of aureochrome 1 from <i>Vaucheria frigida</i> and of GCN4 from <i>S. cerevisiae</i>. The red marked amino acids are involved in the binding of the protein to the ribose phosphate backbone of the target DNA. The blue marked residues interact with the nucleobases <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103307#pone.0103307-Ellenberger1" target="_blank">[14]</a>. The yellow marked arginines interact with both. The sequences show the typical N-X₇-R/K motive with the hepta-repeat of leucines (colored in green) positioned exactly nine amino acids toward the C-terminus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103307#pone.0103307-Jakoby1" target="_blank">[17]</a>.</p

FTIR difference spectrum of aureochrome 1 in the region of 2500 to 2600⁻¹.

<p>Light-dark difference spectra of aureochrome 1 (upper line) and full-length YtvA (lower line) from <i>Bacillus Subtilis</i> in the region between 2500 and 2600 cm⁻¹<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103307#pone.0103307-Bednarz1" target="_blank">[26]</a>. The negative bands correspond to the S-H stretching vibration of a cysteine in the dark state which is lost upon formation of the adduct state (C254 in aureochrome 1 and C62 in YtvA).</p

Kinetic of the ground state recovery.

<p>A) Absorption spectra of aureochrome 1 recorded during the recovery of the dark state. The sample was illuminated with blue-light and spectra were taken at intervals of 45 s. The arrows indicate the increase and decrease of the maxima of the LOV₄₄₅ and LOV₃₉₀ states, respectively. B) Kinetics of recovery of the absorbance at 446 nm of aureochrome 1 after 5 s blue light illumination. The continuous line represents a single exponential fit to the data (dots). The time constant for the ground state recovery was determined to 22±1 min.</p

Kinetic of the triplet state decay.

<p>Kinetics of the absorbance changes at 715(absorption band of the triplet state) of aureochrome 1 after a 10 ns laser flash. Three recordings with a time delay of 30 min after each laser flash have been averaged. The continuous line is the single exponential fit to the data yielding a time constant of τ = 1.5±0.1 µs.</p

Disc Antenna Enhanced Infrared Spectroscopy: From Self-Assembled Monolayers to Membrane Proteins

Plasmonic surfaces have emerged as a powerful platform for biomolecular sensing applications and can be designed to optimize the plasmonic resonance for probing molecular vibrations at utmost sensitivity. Here, we present a facile procedure to generate metallic microdisc antenna arrays that are employed in surface-enhanced infrared absorption (SEIRA) spectroscopy of biomolecules. Transmission electron microscopy (TEM) grids are used as shadow mask deployed during physical vapor deposition of gold. The resulting disc-shaped antennas exhibit enhancement factors of the vibrational bands of 4 × 10⁴ giving rise to a detection limit <1 femtomol (10^–15 mol) of molecules. Surface-bound monolayers of 4-mercaptobenzoic acid show polyelectrolyte behavior when titrated with cations in the aqueous medium. Conformational rigidity of the self-assembled monolayer is validated by density functional theory calculations. The membrane protein sensory rhodopsin II is tethered to the disc antenna arrays and is fully functional as inferred from the light-induced SEIRA difference spectra. As an advance to previous studies, the accessible frequency range is improved and extended into the fingerprint region