Vibrational Spectroscopy
Raman Spectroscopy
Thermal energy allows atoms within molecules to move from their equilibrium position and vibrate.
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Raman spectroscopy probes these molecular vibrations, providing information about the structure, symmetry, electronic environment and bonding. The unique Raman spectral fingerprint can help us identify unknown molecules; it is also highly sensitive to small changes of structural properties of molecules. Raman based imaging, also known as chemical or label free imaging, allows for 2D mapping and 3D imaging of a sample. The images are reconstructed from specific features of Raman spectra, revealing distribution of selected chemical component across the sample (e.g. lipids and proteins in cells).
Resonance Raman (RR) Spectroscopy
Raman intensities are enhanced by several orders of magnitude when excited into electronic transition of the molecule. Only the modes that are coupled to the electronic transition are resonance enhanced, allowing us to selectively probe specific parts of molecules, such as metal (active) sites in metalloenzymes.
By tuning the excitation wavelength, it is possible to observe different chromophores within the same molecule (e.g. 2Fe-2S, 3Fe-4S and 4Fe-4S clusters).
RR spectra of heme proteins are particular informative; the high-frequency region reveals bands that are sensitive to the redox and spin state and coordination pattern of the heme iron.
In the low-frequency region, spectra contain heme Fe-axial ligand stretching and bending modes, allowing for identification of the physiological ligand, or monitoring of substrate binding and formation of transient catalytic intermediate species.
Infrared Spectroscopy
Infrared spectroscopy also probes the vibrational properties of molecules. Due to distinct selection rules, IR active modes are typically different than the Raman modes. In the case of proteins, IR allows us to probe Amide I and Amide II bands arising from backbone amide groups, which are sensitive to secondary structure of proteins. IR can be performed in two different configurations: transmission or attenuated total reflection (ATR). In the latter case, an optical active crystal generates an evanescence wave once invested by infrared beam, it extends into the sample, probing 0.5–2 µm depth .
Surface enhanced Raman (SER) and surface enhanced RR (SERR) spectroscopy
Surface enhanced Raman (SER) spectroscopy takes advantage of the Raman signal enhancement of molecules found in the close proximity to plasmonic (bulk or nanoparticle) metal surfaces. The most commonly used SER-active metals are Ag and Au, which are excited with lasers wavelengths above ca. 320 and 570 nm, respectively.
SER coupled to RR results in Surface Enhanced Resonance Raman spectroscopy, SERRS, (SER + RR → SERR). SERR condition is achieved by using a laser excitation which is simultaneously in resonance with molecular electronic transitions and energy of the surface plasmons; in the case of e.g. heme or FeS metalloproteins, upon immobilization on Ag substrates and excitation with 413 nm. SERRS selectively enhances vibrational modes of the chromophore, providing, in the case of heme proteins, sensitive information on the spin, oxidation and coordination state of the heme group of the adsorbed molecules.
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SERR active surface can serve as a working electrode in SERR spectro-electrochemical experiments, which probe the nature of redox active species and potential dependent redox processes of the immobilized species. Heme proteins have distinct (SE)RR fingerprints in oxidized (Fe3+) and reduced (Fe2+) states, allowing for monitoring redox transitions of the immobilized protein, as well as the potential-dependent changes of spin and coordination state of the heme group.
SERR spectro-electrochemistry can be performed in the TR mode to probe the dynamics of ET processes in the micro- to milli-second range, triggered by a rapid potential jump.
Surface-enhanced infrared absorption (SEIRA) spectroscopy
Surface-enhanced infrared absorption (SEIRA) spectroscopy selectively enhances vibrational modes of the immobilized molecules that undergo the change of dipole moment perpendicular to the surface. In the case of proteins, SEIRA spectra are sensitive to the orientation of the helices with respect to the metal surface, which is reflected in the intensity ratio of amide I and amide II bands. When SEIRA active surface serves as a working electrode, SEIRA spectro-electrochemistry probes potential dependent processes that involve changes of amide I and amide II bands.
Electrochemistry
We use electrochemical techniques to investigate electron transfer (ET) and catalytic properties of metalloproteins, and for the development of enzyme based biosensors. The protein is adsorbed on biocompatible electrode surface and direct electron exchange between the two is monitored. By controlling the driving force for the redox reaction by varying the electrode potentials, and monitoring the current response, thermodynamic (E0) and kinetic (kET rates) information about the redox cofactors are determined simultaneously.
Electrochemical monitoring of the enzyme activity in the presence of a substrate, i.e. electrocatalysis, measures electric currents, which are directly related to the catalytic rates. They originate from redox transformation of substrate into product by the immobilized enzyme, where the electrode acts as a redox partner by transferring electrons to regenerate the active oxidation state. The mechanisms of catalysis can be investigated in the potential domain under enzyme turnover conditions.
Spectro-electrochemistry
Spectro-electrochemistry offers structural fingerprint to electrochemical species. Spectroscopic and electrochemical information are measured simultaneously in situ in electrochemical cells, revealing unique insights into interfacial reactions and redox processes.
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We combine electronic absorption and surface enhanced vibrational spectroscopies with electrochemistry to study potential-dependent processes of metalloproteins immobilized on biocompatible electrodes. Surface-enhanced resonance Raman (SERR) and infrared absorption (SEIRA) spectroscopies are used to characterize the structure, function and thermodynamics of immobilized proteins, to control their orientation and to monitor interactions with substrates and physiological redox partners.
