Kenichi Ataka

Surface-Enhanced Infrared Spectroscopy: The Origin of the Absorption Enhancement and Band Selection Rule in the Infrared Spectra of Molecules Adsorbed on Fine Metal Particles

Infrared transmission spectra of molecules adsorbed on silver island films evaporated on CaF2 have been investigated. The spectra are remarkably simple compared with those of the molecules in the solid state (KBr pellets). Only the vibrational modes which give dipole changes perpendicular to the metal surface are infrared active. In addition, their intensities are about 200 times larger than those of the free molecule. These results can be fully accounted for if the electric field which excites the surface molecule is perpendicular to the local surface of the metal islands and is stronger than the incident electric field. The origin of the absorption enhancement and the surface selection rule is discussed theoretically by using a classical electromagnetic model.

Proteorhodopsin is a Light-driven Proton Pump with Variable Vectoriality

Proteorhodopsin, a homologue of archaeal bacteriorhodopsin (BR), belongs to a newly identified family of retinal proteins from marine bacteria, which could play an important role in the energy balance of the biosphere. We cloned the cDNA sequence of proteorhodopsin by chemical gene synthesis, expressed the protein in Escherichia coli cells, purified and reconstituted the protein in its functional active state. The photocycle characteristics were determined by time-resolved absorption and Fourier transform infrared (FT-IR) spectroscopy. The pH-dependence of the absorption spectrum indicates that the pK(a) of the primary acceptor of the Schiff base proton (Asp97) is 7.68. Generally, the photocycle of proteorhodopsin is similar to that of BR, although an L-like photocycle intermediate was not detectable. Whereas at pH>7 an M-like intermediate is formed upon illumination, at pH 5 no M-like intermediate could be detected. As the photocycle kinetics do not change between the acidic and alkaline state of proteorhodopsin, the only difference between these two forms is the protonation status of Asp97. This is corroborated by time-resolved FT-IR spectroscopy, which demonstrates that proton transfer from the retinal Schiff base to Asp97 is observed at alkaline pH, but the other vibrational changes are essentially pH-independent.After reconstitution into proteoliposomes, light-induced proton currents of proteorhodopsin were measured in a compound membrane system where proteoliposomes were adsorbed to planar lipid bilayers. Our results show that proteorhodopsin is a light-driven proton pump with characteristics similar to those of BR at alkaline pH. However, at acidic pH, the direction of proton pumping is inverted. Complementary experiments were carried out on proteorhodopsin expressed heterologously in Xenopus laevis oocytes under voltage clamp conditions. The following results were obtained. (1) At alkaline pH, proteorhodopsin mediates outwardly directed proton pumping like BR. (2) The direction of proton pumping can be inverted, when Asp97 is protonated. (3) The current can be inverted by changes of the polarity of the applied voltage. (4) The light intensity-dependence of the photocurrents leads to the conclusion that the alkaline form of proteorhodopsin shows efficient proton pumping after sequential excitation by two photons.

Photosynthetic Hydrogen Production by a Hybrid Complex of Photosystem I and [NiFe]-Hydrogenase

Nature provides key components for generating fuels from renewable resources in the form of enzymatic nanomachines which catalyze crucial steps in biological energy conversion, for example, the photosynthetic apparatus, which transforms solar power into chemical energy, and hydrogenases, capable of generating molecular hydrogen. As sunlight is usually used to synthesize carbohydrates, direct generation of hydrogen from light represents an exception in nature. On the molecular level, the crucial step for conversion of solar energy into H(2) lies in the efficient electronic coupling of photosystem I and hydrogenase. Here we show the stepwise assembly of a hybrid complex consisting of photosystem I and hydrogenase on a solid gold surface. This device gave rise to light-induced H(2) evolution. Hydrogen production is possible at far higher potential and thus lower energy compared to those of previously described (bio)nanoelectronic devices that did not employ the photosynthesis apparatus. The successful demonstration of efficient solar-to-hydrogen conversion may serve as a blueprint for the establishment of this system in a living organism with the paramount advantage of self-replication.

Biochemical applications of surface-enhanced infrared absorption spectroscopy

AbstractAn overview is presented on the application of surface-enhanced infrared absorption (SEIRA) spectroscopy to biochemical problems. Use of SEIRA results in high surface sensitivity by enhancing the signal of the adsorbed molecule by approximately two orders of magnitude and has the potential to enable new studies, from fundamental aspects to applied sciences. This report surveys studies of DNA and nucleic acid adsorption to gold surfaces, development of immunoassays, electron transfer between metal electrodes and proteins, and protein–protein interactions. Because signal enhancement in SEIRA uses surface properties of the nano-structured metal, the biomaterial must be tethered to the metal without hampering its functionality. Because many biochemical reactions proceed vectorially, their functionality depends on proper orientation of the biomaterial. Thus, surface-modification techniques are addressed that enable control of the proper orientation of proteins on the metal surface. FigureSurface enhanced infrared absorption spectroscopy (SEIRAS) on the studies of tethered protein monolayer (cytochrome c oxidase and cytochrome c) on gold substrate (left), and its potential induced surface enhanced infrared difference absorption (SEIDA) spectrum

Resolving voltage-dependent structural changes of a membrane photoreceptor by surface-enhanced IR difference spectroscopy

Membrane proteins are molecular machines that transport ions, solutes, or information across the cell membrane. Electrophysiological techniques have unraveled many functional aspects of ion channels but suffer from the lack of structural sensitivity. Here, we present spectroelectrochemical data on vibrational changes of membrane proteins derived from a single monolayer. For the seven-helical transmembrane protein sensory rhodopsin II, structural changes of the protein backbone and the retinal cofactor as well as single ion transfer events are resolved by surface-enhanced IR difference absorption spectroscopy (SEIDAS). Angular changes of bonds versus the membrane normal have been determined because SEIDAS monitors only those vibrations whose dipole moment are oriented perpendicular to the solid surface. The application of negative membrane potentials (ΔV = −0.3 V) leads to the selective halt of the light-induced proton transfer at the stage of D75, the counter ion of the retinal Schiff base. It is inferred that the voltage raises the energy barrier of this particular proton-transfer reaction, rendering the energy deposited in the retinal by light excitation insufficient for charge transfer to occur. The other structural rearrangements that accompany light-induced activity of the membrane protein, are essentially unaffected by the transmembrane electric field. Our results demonstrate that SEIDAS is a generic approach to study processes that depend on the membrane potential, like those in voltage-gated ion channels and transporters, to elucidate the mechanism of ion transfer with unprecedented spatial sensitivity and temporal resolution.

Thinner, Smaller, Faster: IR Techniques To Probe the Functionality of Biological and Biomimetic Systems

New techniques in vibrational spectroscopy are promising for the study of biological samples as they provide exquisite spatial and/or temporal resolution with the benefit of minimal perturbation of the system during observation. In this Minireview we showcase the power of modern infrared techniques when applied to biological and biomimetic systems. Examples will be presented on how conformational changes in peptides can be traced with femtosecond resolution and nanometer sensitivity by 2D IR spectroscopy, and how surface-enhanced infrared difference absorption spectroscopy can be used to monitor the effect of the membrane potential on a single proton-transfer step in an integral membrane protein. Vibrational spectra of monolayers of molecules at basically any interface can be recorded with sum-frequency generation, which is strictly surface-sensitive. Chemical images are recorded by applying scanning near-field infrared microscopy at lateral resolutions better than 50 nm.

Surface-enhanced infrared absorption spectroscopy (SEIRAS) to probe monolayers of membrane proteins.

Surface-enhanced infrared absorption spectroscopy (SEIRAS) represents a variation of conventional infrared spectroscopy and exploits the signal enhancement exerted by the plasmon resonance of nano-structured metal thin films. The surface enhancement decays in about 10nm with the distance from the surface and is, thus, perfectly suited to selectively probe monolayers of biomembranes. Peculiar to membrane proteins is their vectorial functionality, the probing of which requires proper orientation within the membrane. To this end, the metal surface used in SEIRAS is chemically modified to generate an oriented membrane protein film. Monolayers of uniformly oriented membrane proteins are formed by tethering His-tagged proteins to a nickel nitrilo-triacetic acid (Ni-NTA) modified gold surface and SEIRAS commands molecular sensitivity to probe each step of surface modification. The solid surface used as plasmonic substrate for SEIRAS, can also be employed as an electrode to investigate systems where electron transfer reactions are relevant, like e.g. cytochrome c oxidase or plant-type photosystems. Furthermore, the interaction of these membrane proteins with water-soluble proteins, like cytochrome c or hydrogenase, is studied on the molecular level by SEIRAS. The impact of the membrane potential on protein functionality is verified by monitoring light-dark difference spectra of a monolayer of sensory rhodopsin (SRII) at different applied potentials. It is demonstrated that the interpretations of all of these experiments critically depend on the orientation of the solid-supported membrane protein. Finally, future directions of SEIRAS including cellular systems are discussed. This article is part of a Special Issue entitled: FTIR in membrane proteins and peptide studies.

Immobilization of the (FeFe)-hydrogenase CrHydA1 on a gold electrode: Design of a catalytic surface for the production of molecular hydrogen

Hydrogenase-modified electrodes are a promising catalytic surface for the electrolysis of water with an overpotential close to zero. The [FeFe]-hydrogenase CrHydA1 from the photosynthetic green alga Chlamydomonas reinhardtii is the smallest [FeFe]-hydrogenase known and exhibits an extraordinary high hydrogen evolution activity. For the first time, we immobilized CrHydA1 on a gold surface which was modified by different carboxy-terminated self-assembled monolayers. The immobilization was in situ monitored by surface-enhanced infrared spectroscopy. In the presence of the electron mediator methyl viologen the electron transfer from the electrode to the hydrogenase was detected by cyclic voltammetry. The hydrogen evolution potential (-290 mV vs NHE, pH 6.8) of this protein modified electrode is close to the value for bare platinum (-270 mV vs NHE). The surface coverage by CrHydA1 was determined to 2.25 ng mm(-2) by surface plasmon resonance, which is consistent with the formation of a protein monolayer. Hydrogen evolution was quantified by gas chromatography and the specific hydrogen evolution activity of surface-bound CrHydA1 was calculated to 1.3 micromol H(2)min(-1)mg(-1) (or 85 mol H(2)min(-1)mol(-1)). In conclusion, a viable hydrogen-evolving surface was developed that may be employed in combination with immobilized photosystems to provide a platform for hydrogen production from water and solar energy with enzymes as catalysts.

Reconstitution of [Fe]-hydrogenase using model complexes

[Fe]-Hydrogenase catalyses the reversible hydrogenation of a methenyltetrahydromethanopterin substrate, which is an intermediate step during the methanogenesis from CO2 and H2. The active site contains an iron-guanylylpyridinol cofactor, in which Fe(2+) is coordinated by two CO ligands, as well as an acyl carbon atom and a pyridinyl nitrogen atom from a 3,4,5,6-substituted 2-pyridinol ligand. However, the mechanism of H2 activation by [Fe]-hydrogenase is unclear. Here we report the reconstitution of [Fe]-hydrogenase from an apoenzyme using two FeGP cofactor mimics to create semisynthetic enzymes. The small-molecule mimics reproduce the ligand environment of the active site, but are inactive towards H2 binding and activation on their own. We show that reconstituting the enzyme using a mimic that contains a 2-hydroxypyridine group restores activity, whereas an analogous enzyme with a 2-methoxypyridine complex was essentially inactive. These findings, together with density functional theory computations, support a mechanism in which the 2-hydroxy group is deprotonated before it serves as an internal base for heterolytic H2 cleavage.

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.