Surface-enhanced resonance Raman scattering (SERRS) of rhodamine 6G (R6G) adsorbed on colloidal silver was studied. Adsorption isotherms could be obtained from SERRS and fluorescent measurements. Two different kinds of adsorption sites were inferred from the isotherms. One kind is rather unspecific and shows a high surface coverage. The enhancement factor at such sites is 3000, which can be well explained by the classical electromagnetic theory of colloids. The second kind is only observed in the presence of anions (Cl-, I-, Br-, F, SO:-). Specific active sites are formed at an extremely low surface coverage. It was concluded from the isotherms that at such sites the molecules are chemisorbed. Overall enhancement factors up to lo6 were found for molecules at anion-activated sites. The additional enhancement factor is ascribed to a local mechanism of an R6G-adatom (or cluster)-anion surface complex. SERRS excitation profiles of active sites are closely related to the molecular resonance at 530 nm. SERRS spectra of R6G were recorded and analyzed in a wide frequency range. The two adsorption sites could be distinguished by characteristic vibrational features. It was demonstrated that SERRS is also a powerful analytical tool for dye molecules. IntroductionThe discovery of the surface-enhanced Raman (SER) effect of pyridine adsorbed on a roughened silver electrode has opened a wide research field both in physics and chemistry of interfaces and in Raman spectro~copy.'-~ By now a large number of experimental data have been accumulated. However, a generally accepted and uniform theoretical explanation of this exciting phenomenon has not been established." Two different theoretical approaches are mainly d i s c~s s e d .~The first one uses the plasma resonance model, which is related to the optical properties of free-electron-like metals.@ Irradiation of a microscopically rough metal surface gives rise to local plasma modes. The electric field at such a surface becomes very large if the incident photon energy is in resonance with a normal mode of the conduction electrons in the metal. This leads to enhanced Raman scattering from those molecules which are close to the metal surface.The second approach is based on the concept of "active sites" at the metal surface.'w12 A specific interaction between the metal and the molecules occupying these sites induces enhanced Raman scattering. The nature of this interaction is controversial. Some investigators have suggested that charge-transfer processes are involved between the metal and the molecule via the active sites, which are supposed to be metal adatoms or clusters. This model implies that Raman enhancement is restricted to molecules immediately adjacent to the metal while the electromagnetic approach predicts an enhancement also for molecules at a distance

Protein and heme structural changes of ferric and ferrous cytochrome c (Cyt-c) that are induced by electrostatic binding (e.g., liposomes, electrodes), by hydrophobic interactions (e.g., monomeric sodium dodecyl sulfate), by guanidium hydrochloride (GuHCl), and at low pH and high temperature were studied by UV-vis absorption, circular dichroism (CD), electron paramagnetic resonance (EPR), and (surface-enhanced) resonance Raman [(SE)RR] spectroscopy. In a global spectral analysis, all species that differ with respect to the heme structure were identified and characterized in terms of the spin and ligation state of the heme as well as of protein secondary and tertiary structure changes. The results indicate that the upper part of the heme pocket including the Met-80 ligand is the most labile protein region such that this ligand is dissociated from the heme iron in all nonnative Cyt-c states. Among these states, there are two six-coordinated low-spin (LS) configurations with H 2 O or His-33 serving as the sixth (axial) ligand. Whereas the ferric H 2 O/His-18-ligated low-spin species is only formed in the A state at low pH and high ionic strength, the His-33/His-18-ligation pattern corresponds to a stable ferric configuration inasmuch as it can be induced by electrostatic and hydrophobic interactions and under nondenaturing and denaturing conditions, that is, nearly independent of the secondary structure. Conversely, the heme pocket on the opposite side of the heme remains largely preserved except for ferric Cyt-c at very low pH and high GuHCl concentrations as indicated by the replacement of His-18 by a water molecule. Structural changes that are localized in the heme pocket and lead to a ferric bis-His-coordinated LS, a ferric water/His and mono-His high-spin (HS), and a ferrous mono-His HS configuration may be induced by hydrophobic or electrostatic interactions with the front surface of Cyt-c. The present study contributes to a consistent description of the conformational manifold of Cyt-c, which is essential for elucidating the role of conformational transitions during the natural functions of Cyt-c in energy transduction and apoptosis.

Cytochrome c (Cyt-c) was electrostatically bound to self-assembled monolayers (SAM) of ω-carboxylalkanethiols that were covalently attached to Ag electrodes. Employing surface-enhanced resonance Raman (SERR) spectroscopy, the redox equilibria and the structural changes of the adsorbed Cyt-c were analyzed quantitatively for SAMs of different chain lengths ranging from 2-mercaptoacetic acid (C 2 -SAM) to 16mercaptohexadecanoic acid (C 16 -SAM). In the presence of Cyt-c in the bulk solution, the SERR spectra of the adsorbed Cyt-c display the characteristic vibrational band pattern of the native protein conformation denoted as state B1. The enhancement of the SERR signals decreases with increasing chain length, but even at distances as large as 24 Å (C 16 -SAM), SERR spectra of high quality could be obtained. Conversely, no SERR signals could be detected for SAMs including hydroxyl instead of carboxylate headgroups, implying that Cyt-c is adsorbed via electrostatic interactions. On the basis of potential-dependent SERR experiments, the redox equilibria of the adsorbed Cyt-c (B1) were analyzed, revealing ideal Nernstian behavior (n = 1). However, the redox potentials exhibit negative shifts compared to that of Cyt-c in solution, which increase with the chain length of the SAMs. In the absence of excess Cyt-c in solution (i.e., 0.2 µM), a new conformational state B2 of the adsorbed Cyt-c is observed. This state B2, which differs from the native state B1 by the heme pocket structure, includes three substates of different spin and coordination configurations. The distribution among these substates as well as the total contribution of state B2 varies with the chain length of the SAM such that the latter decreases from 73% at C 2 -SAM to 0% at C 11 -and C 16 -SAMs. These results imply that the formation of B2 is induced by the electric field at the binding site, generated by the potential drop across the electrode/SAM interface. When an electrostatic model for the interfacial potential distribution for the electrode/ SAM/protein device is employed, both the redox potential shifts and the electric-field-induced structural changes can be consistently explained. The impact of these findings for the processes of Cyt-c at biological interfaces is discussed.

The ability of phytochromes (Phy) to act as photointerconvertible light switches in plants and microorganisms depends on key interactions between the bilin chromophore and the apoprotein that promote bilin attachment and photointerconversion between the spectrally distinct red light-absorbing Pr conformer and far red light-absorbing Pfr conformer. Using structurally guided site-directed mutagenesis combined with several spectroscopic methods, we examined the roles of conserved amino acids within the bilin-binding domain of Deinococcus radiodurans bacteriophytochrome with respect to chromophore ligation and Pr/Pfr photoconversion. Incorporation of biliverdin IX␣ (BV), its structure in the Pr state, and its ability to photoisomerize to the first photocycle intermediate are insensitive to most single mutations, implying that these properties are robust with respect to small structural/electrostatic alterations in the binding pocket. In contrast, photoconversion to Pfr is highly sensitive to the chromophore environment. Many of the variants form spectrally bleached Meta-type intermediates in red light that do not relax to Pfr. Particularly important are Asp-207 and His-260, which are invariant within the Phy superfamily and participate in a unique hydrogen bond matrix involving the A, B, and C pyrrole ring nitrogens of BV and their associated pyrrole water. Resonance Raman spectroscopy demonstrates that substitutions of these residues disrupt the Pr to Pfr protonation cycle of BV with the chromophore locked in a deprotonated Meta-R c -like photoconversion intermediate after red light irradiation. Collectively, the data show that a number of contacts contribute to the unique photochromicity of Phy-type photoreceptors. These include residues that fix the bilin in the pocket, coordinate the pyrrole water, and possibly promote the proton exchange cycle during photoconversion.The phytochrome (Phy) 5 superfamily encompasses a large and diverse set of photoreceptors present in the plant, fungal, and bacterial kingdoms where they play critical roles in various light-regulated processes (1-3). These processes range from the control of phototaxis, pigmentation, and photosynthetic potential in proteobacteria and cyanobacteria to seed germination, chloroplast development, shade avoidance, and flowering time in higher plants. Phys are unique among photoreceptors in being able to assume two stable, photointerconvertible conformers, designated Pr and Pfr based on their respective absorption maxima in the red and far-red spectral regions. By cycling between Pr and Pfr, Phys act as light-regulated switches in various photosensory cascades.Phys are homodimeric complexes with each polypeptide containing a single bilin (or linear tetrapyrrole) chromophore, which binds autocatalytically via a thioether linkage to a positionally conserved cysteine (1-3). The photosensing portion typically contains Per/Arndt/Sim (PAS) and cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) domains, which are essential for bilin binding and Pr assembly, and t...

Cytochrome c (Cyt-c) was electrostatically bound to self-assembled monolayers (SAM) on an Ag electrode, which are formed by omega-carboxyl alkanethiols of different chain lengths (C(x)). The dynamics of the electron-transfer (ET) reaction of the adsorbed heme protein, initiated by a rapid potential jump to the redox potential, was monitored by time-resolved surface enhanced resonance Raman (SERR) spectroscopy. Under conditions of the present experiments, only the reduced and oxidized forms of the native protein state contribute to the SERR spectra. Thus, the data obtained from the spectra were described by a one-step relaxation process yielding the rate constants of the ET between the adsorbed Cyt-c and the electrode for a driving force of zero electronvolts. For C(16)- and C(11)-SAMs, the respective rate constants of 0.073 and 43 s(-1) correspond to an exponential distance dependence of the ET (beta = 1.28 A(-1)), very similar to that observed for long-range intramolecular ET of redox proteins. Upon further decreasing the chain length, the rate constant only slightly increases to 134 s(-1) at C(6)- and remains essentially unchanged at C(3)- and C(2)-SAMs. The onset of the nonexponential distance dependence is paralleled by a kinetic H/D effect that increases from 1.2 at C(6)- to 4.0 at C(2)-coatings, indicating a coupling of the redox reaction with proton-transfer (PT) steps. These PT processes are attributed to the rearrangement of the hydrogen-bonding network of the protein associated with the transition between the oxidized and reduced state of Cyt-c. Since this unusual kinetic behavior has not been observed for electron-transferring proteins in solution, it is concluded that at the Ag/SAM interface the energy barrier for the PT processes of the adsorbed Cyt-c is raised by the electric field. This effect increases upon reducing the distance to the electrode, until nuclear tunneling becomes the rate-limiting step of the redox process. The electric field dependence of the proton-coupled ET may represent a possible mechanism for controlling biological redox reactions via changes of the transmembrane potential.

The heme protein cytochrome c acts as an electron carrier at the mitochondrial-membrane interface and thus exerts its function under the influence of strong electric fields. To assess possible consequences of electric fields on the redox processes of cytochrome c, the protein can be immobilized to self-assembled monolayers on electrodes and studied by surface-enhanced resonance Raman spectroscopy. Such model systems may mimic some essential features of biological interfaces including local electric field strengths. It is shown that physiologically relevant electric field strengths can effectively modulate the electron-transfer dynamics and induce conformational transitions.

The P r 3 P fr phototransformation of the bacteriophytochrome Agp1 from Agrobacterium tumefaciens and the structures of the biliverdin chromophore in the parent states and the cryogenically trapped intermediate Meta-R C were investigated with resonance Raman spectroscopy and flash photolysis. Strong similarities with the resonance Raman spectra of plant phytochrome A indicate that in Agp1 the methine bridge isomerization state of the chromophore is ZZZasa in P r and ZZEssa in P fr , with all pyrrole nitrogens being protonated. Photoexcitation of P r is followed by (at least) three thermal relaxation components in the formation of P fr with time constants of 230 s and 3.1 and 260 ms. H 2 O/D 2 O exchange reveals kinetic isotope effects of 1.9, 2.6, and 1.3 for the respective transitions that are accompanied by changes of the amplitudes. The second and the third relaxation correspond to the formation and decay of Meta-R C , respectively. Resonance Raman measurements of Meta-R C indicate that the chromophore adopts a deprotonated ZZE configuration. Measurements with a pH indicator dye show that formation and decay of Meta-R C are associated with proton release and uptake, respectively. The stoichiometry of the proton release corresponds to one proton per photoconverted molecule. The coupling of transient chromophore deprotonation and proton release, which is likely to be an essential element in the P r 3 P fr photoconversion mechanism of phytochromes in general, may play a crucial role for the structural changes in the final step of the P fr formation that switch between the active and the inactive state of the photoreceptor.Phytochromes are photoreceptors that utilize light as a source of information for controlling numerous biological processes (1, 2). The chromophore, a methine-bridged tetrapyrrole ( Fig. 1), acts as a photoswitch between two stable, spectrally distinct forms, denoted as P r and P fr according to the red and far-red absorption maxima, respectively. The P r /P fr interconversion is initiated by the rapid Z/E photoisomerization of the C-D methine bridge (3), followed by chromophore relaxations that are coupled to structural changes of the apoprotein (4). These structural changes are the trigger for signal transduction. Resonance Raman (RR) 2 and IR spectroscopy have provided valuable insight into light-induced chromophore and protein structural changes (e.g. see Refs. 5-10), but molecular and mechanistic details are not yet known and no crystal structure of a phytochrome has been reported so far.While phytochromes were originally thought to be restricted to plants, the discovery of these chromoproteins in cyanobacteria (11) and other bacteria points to the prokaryotic origin of this family of photoreceptors. In contrast to plant phytochromes, typical bacterial phytochromes are light-regulated histidine kinases. Despite the quite different regulatory functions (12, 13), plant and bacterial phytochromes exhibit structural and mechanistic similarities. The phytochromobilin chromophore of plant phytoc...

The photoconversion of phytochrome (phytochrome A from Avena satina) from the inactive (Pr) to the physiologically active form (Pfr) was studied by near-infrared Fourier transform resonance Raman spectroscopy at cryogenic temperatures, which allow us to trap the intermediate states. Nondeuterated and deuterated buffer solutions were used to determine the effect of H/D exchange on the resonance Raman spectra. For the first time, reliable spectra of the "bleached" intermediates meta-R(A) and meta-R(C) were obtained. The vibrational bands in the region 1300-1700 cm(-)(1), which is particularly indicative of structural changes in tetrapyrroles, were assigned on the basis of recent calculations of the Raman spectra of the chromophore in C-phycocyanin and model compounds [Kneip, C., Hildebrandt, P., Németh, K., Mark, F., Schaffner, K. (1999) Chem. Phys. Lett. 311, 479-485]. The experimental resonance Raman spectra Pr are compatible with the Raman spectra calculated for the protonated ZZZasa configuration, which hence is suggested to be the chromophore structure in this parent state of phytochrome. Furthermore, marker bands could be identified that are of high diagnostic value for monitoring structural changes in individual parts of the chromophore. Specifically, it could be shown that not only in the parent states Pr and Pfr but also in all intermediates the chromophore is protonated at the pyrroleninic nitrogen. The spectral changes observed for lumi-R confirm the view that the photoreaction of Pr is a Z --> E isomerization of the CD methine bridge. The subsequent thermal decay reaction to meta-R(A) includes relaxations of the CD methine bridge double bond, whereas the formation of meta-R(C) is accompanied by structural adaptations of the pyrrole rings B and C in the protein pocket. The far-reaching similarities between the chromophores of meta-R(A) and Pfr suggest that in the step meta-R(A) --> Pfr the ultimate structural changes of the protein matrix occur.