Sub-millisecond Time Domain Research Articles

Synchrotron Infrared Radiation for Electrochemical External Reflection Spectroscopy: A Case Study Using Ferrocyanide

Synchrotron infrared radiation has been successfully coupled through an infrared (IR) microscope to a thin-cavity external reflectance cell to study the diffusion controlled redox of a ferrocyanide solution. Excellent signal-to-noise ratios were achieved even at aperture settings close to the diffraction limit. Comparisons of noise levels as a function of aperture size demonstrate that this can be attributed to the high brilliance of synchrotron radiation relative to a conventional thermal source. Time resolved spectroscopic studies of diffusion controlled redox behavior have been measured and compared to purely electrochemical responses of the thin-cavity cell. Marked differences between the two measurements have been explained by analyzing diffusion in both the axial (linear) and radial dimensions. Whereas both terms contribute to the measured current and charge, only species that originate in the volume element above the electrode and diffuse in the direction perpendicular to the electrode surface are interrogated by IR radiation. Implications for the use of ultramicroelectrodes and synchrotron IR (SIR) to study electrochemical processes in the submillisecond time domain are discussed.

An On-pathway Intermediate in the Folding of a PDZ Domain

The folding pathways of some proteins include the population of partially structured species en route to the native state. Identification and characterization of these folding intermediates are particularly difficult as they are often only transiently populated and play different mechanistic roles, being either on-pathway productive species or off-pathway kinetic traps. To define the role of folding intermediates, a quantitative analysis of the folding and unfolding rate constants over a wide range of denaturant concentration is often required. Such a task is further complicated by the reversible nature of the folding reaction, which implies the observed kinetics to be governed by a complex combination of different microscopic rate constants. Here, we tackled this problem by measuring directly the folding rate constant under highly denaturing conditions, namely by inducing the folding of a PDZ domain through a quasi-irreversible binding reaction with a specific peptide. In analogy with previous works based on hydrogen exchange experiments, we present evidence that the folding pathway of the PDZ domain involves the formation of an obligatory on-pathway intermediate. The results presented exemplify a novel type of kinetic test to detect an on-pathway folding intermediate.

Voltage changes involving photosystem II quinone–iron complex turnover

An electrometrical technique was used to investigate proton-coupled electron transfer between the primary plastoquinone acceptor Q (A) (-) and the oxidized non-heme iron Fe(3+) on the acceptor side of photosystem II core particles incorporated into phospholipid vesicles. The sign of the transmembrane electric potential difference Deltapsi (negative charging of the proteoliposome interior) indicates that the iron-quinone complex faces the interior surface of the proteoliposome membrane. Preoxidation of the non-heme iron was achieved by addition of potassium ferricyanide entrapped into proteoliposomes. Besides the fast unresolvable kinetic phase (tau approximately 0.1 micro s) of Deltapsi generation related to electron transfer between the redox-active tyrosine Y(Z) and Q(A), an additional phase in the submillisecond time domain (tau approximately 0.1 ms at 23 degrees C, pH 7.0) and relative amplitude approximately 20% of the amplitude of the fast phase was observed under exposure to the first flash. This phase was absent under the second laser flash, as well as upon the first flash in the presence of DCMU, an inhibitor of electron transfer between Q(A) and the secondary quinone Q(B). The rate of the additional electrogenic phase is decreased by about one-half in the presence of D(2)O and is reduced with the temperature decrease. On the basis of the above observations we suggest that the submillisecond electrogenic reaction induced by the first flash is due to the vectorial transfer of a proton from external aqueous phase to an amino acid residue(s) in the vicinity of the non-heme iron. The possible role of the non-heme iron in cyclic electron transfer in photosystem II complex is discussed.

Electro-optical studies on MDMO-PPV:PCBM bulk-heterojunction solar cells on the millisecond time scale: Trapped carriers

Abstract The influence of a periodically modulated electric field on the concentration of photogenerated charges in bulk-heterojunction solar cells of poly[2-methoxy-5-(3′,7′-dimethyloctyloxy)-1,4-phenylene vinylene] (MDMO-PPV) and 1-(3-methoxycarbonyl)-propyl-1-phenyl-[6,6]C61 (PCBM) is studied at 80 K by near-steady-state photoinduced absorption (PIA) spectroscopy. Measurements are complemented with recording the photocurrent. The results show that the lifetime of extractable charge carriers under conditions where the built-in electrical field is largely compensated, is in the sub-millisecond time domain. PIA is dominated by the contribution from trapped carriers and application of a −4 V bias increases the number of trap sites available to the carriers in comparison with a +1 V bias voltage. The reverse bias voltage also leads to an enhancement in the generation rate of carriers that can be trapped and/or the lifetime of the trapped carriers. The experiment indicates that the lifetime of the trapped carriers at −4 V bias voltage remains in the sub-millisecond time domain.

Microwave enhanced electrochemistry: mass transport effects and steady state voltammetry in the sub-millisecond time domain

In situ microwave activation of electrochemical processes is possible by self-focusing of intense microwave radiation into a region close to the electrode|solution (electrolyte) interface of a microelectrode placed inside a microwave cavity. A systematic study of the microwave activation effects in electrochemical processes is reported for two redox systems, Fe ( CN ) 6 3 - / 4 - and Ru ( NH 3 ) 6 3 + / 2 + , in aqueous KCl solution. Platinum microelectrodes of 100, 50, and 25 μm diameter are employed and at the 25 μm diameter electrode, extreme current enhancements of up to three orders of magnitude are detected. A typical Nernst diffusion layer thickness in aqueous solution of less than 100 nm can be achieved routinely and, consequently, high temperature steady state voltammetry is possible in the sub-millisecond time domain. Volatile reagents reduce the efficiency of this effect and therefore a steam bubble mechanism is proposed to explain the observations. Microwave effects on the rate of interfacial electron transfer are discussed.

Microwave enhanced electrochemistry: mass transport effects and steady state voltammetry in the sub-millisecond time domain

In situ microwave activation of electrochemical processes is possible by self-focusing of intense microwave radiation into a region close to the electrode|solution (electrolyte) interface of a microelectrode placed inside a microwave cavity. A systematic study of the microwave activation effects in electrochemical processes is reported for two redox systems, Fe ( CN ) 6 3 - / 4 - and Ru ( NH 3 ) 6 3 + / 2 + , in aqueous KCl solution. Platinum microelectrodes of 100, 50, and 25 μm diameter are employed and at the 25 μm diameter electrode, extreme current enhancements of up to three orders of magnitude are detected. A typical Nernst diffusion layer thickness in aqueous solution of less than 100 nm can be achieved routinely and, consequently, high temperature steady state voltammetry is possible in the sub-millisecond time domain. Volatile reagents reduce the efficiency of this effect and therefore a steam bubble mechanism is proposed to explain the observations. Microwave effects on the rate of interfacial electron transfer are discussed.

Collapse and search dynamics of apomyoglobin folding revealed by submillisecond observations of alpha-helical content and compactness.

The characterization of protein folding dynamics in terms of secondary and tertiary structures is important in elucidating the features of intraprotein interactions that lead to specific folded structures. Apomyoglobin (apoMb), possessing seven helices termed A-E, G, and H in the native state, has a folding intermediate composed of the A, G, and H helices, whose formation in the submillisecond time domain has not been clearly characterized. In this study, we used a rapid-mixing device combined with circular dichroism and small-angle x-ray scattering to observe the submillisecond folding dynamics of apoMb in terms of helical content (f(H)) and radius of gyration (R(g)), respectively. The folding of apoMb from the acid-unfolded state at pH 2.2 was initiated by a pH jump to 6.0. A significant collapse, corresponding to approximately 50% of the overall change in R(g) from the unfolded to native conformation, was observed within 300 micros after the pH jump. The collapsed intermediate has a f(H) of 33% and a globular shape that involves >80% of all its atoms. Subsequently, a stepwise helix formation was detected, which was interpreted to be associated with a conformational search for the correct tertiary contacts. The characterized folding dynamics of apoMb indicates the importance of the initial collapse event, which is suggested to facilitate the subsequent conformational search and the helix formation leading to the native structure.

Insights from protein film voltammetry into mechanisms of complex biological electron-transfer reactionsBased on the presentation given at Dalton Discussion No. 4, 10–13th January 2002, Kloster Banz, Germany.

As the fundamental principles of long-range electron transfer (ET) in proteins are becoming well understood, interest is focusing on the mechanisms by which ET is coupled to chemical reactions such as ion transport and catalysis. Protein film voltammetry provides a powerful way to investigate these problems. The protein is immobilised on an electrode as an adsorbed electroactive film, typically a monolayer or less: then, by applying a potential, electrons are driven in and out of the active sites, resulting in diagnostically useful current signals. It is possible to resolve complex reactions over a wide dynamic range. For example, with cyclic voltammetry, scan rates exceeding 1000 V s−1 can be used to observe coupling reactions that occur in the sub-millisecond time domain. For enzymes, catalysis can be measured as a function of potential to reveal processes that are mechanistically informative and may be involved in controlling activity. This paper will illustrate the capabilities of this approach for mechanistic investigations into biological redox chemistry.

Protein Film Voltammetry: Revealing the Mechanisms of Biological Oxidation and Reduction

One of the most intriguing and important aspects of biological chemistry is how the deceptively simple process of electron transfer (ET) is utilized and organized by macromolecules that perform some of the most sophisticated chemistry known to man. The fundamental principles of ET are now fairly well understood, and interest is being focused instead on the mechanisms by which ET in proteins is coupled to chemical reactions such as ion transport and catalysis. Protein film voltammetry provides a powerful way to investigate these problems. The protein is immobilized on an electrode as an absorbed electroactive film and by applying a potential, electrons are driven in and out of the active sites. Signals are obtained from extremely small sample quantities (monolayer coverage or less), and from these it is possible to detect and characterize active sites and to resolve complex reactions. Experiments may be carried out over a wide dynamic range: for example, with cyclic voltammetry scan rates exceeding 1000 V/s can be used to observe coupling reactions that occur in the sub-millisecond time domain. For enzymes, fast cycles can be used to “trap” intermediates; alternatively steady-state catalysis and redox-linked activation can be studied using slow scan rates or potential step methods. This paper explains the concept of protein film voltammetry and illustrates how it can be applied to some complex problems in biological redox chemistry.

Photochemistry of Photochromic Benzopyrans Studied by Time-Resolved Absorption Spectroscopy

Photochromic reactions of 2H-benzopyrans have been extensively studied to design new materials for commercial applications such as optical memories. Photochromism of 2H-benzopyrans proceeds from a C–O bond cleavage of the colorless closed form to give the colored open forms, which can thermally revert back to the original closed form. From time-resolved absorption spectroscopy, the ring opening of 2H-benzopyrans, 2,4-diphenyl-and 2,2,4-triphenyl-2H-benzopyran, is found to occur via the excited singlet state within 2 ps to produce vibrationally excited open forms in the ground electronic state. In the subnanosecond to submillisecond time domain, several decay components are observed. These components are assigned to respective stereoisomers with respect to two double bonds and one single bond of the open enone forms. As revealed from pump-laser power dependencies of the yields of the open forms, the photocleavage of the benzopyran molecules gives at first only the open forms revertible to the closed form by a single-bond rotation, and the photoexcitation of the first generated open forms gives rise to other open forms that need a double-bond rotation for reversion to the original closed form. Such a two-step two-photon photochromism can be expected to have a wide range of application.

Time-Resolved Absorption Studies on the Photochromic Process of 2H-Benzopyrans in the Picosecond to Submillisecond Time Domain

Picosecond to submillisecond photochromic reactions of 2,4-diphenyl-2H-benzopyran and 2,2,4-triphenyl-2H-benzopyran have been investigated by time-resolved absorption spectroscopy. The C−O bond cleavage of the benzopyrans (closed forms) occurs via the first excited singlet state within 2 ps to produce vibrationally excited open forms in the ground electronic state. In the subnanosecond to submillisecond time domain, several decay components with almost the same spectral profiles are observed. These components are assigned to respective stereoisomers with respect to two double bonds and one single bond of the open enone forms. From the pump-laser power dependencies of the yields of the open forms, it is suggested that the photocleavage gives at first only the open forms revertible to the closed form by a single-bond rotation, and that the photoexcitation of the first generated open forms gives rise to other open forms which need a double-bond rotation for reversion to the closed form. The photochromic reactions of a series of 2H-benzopyrans bearing substituents on the pyran ring have also been studied using nanosecond time-resolved absorption spectroscopy. The size of a substituent in the 4-position fairly affects the rate constants of the thermal reversion of the open form to the closed form.

Myosin head rotation in muscle fibers measured using polarized fluorescence photobleaching recovery

The technique of polarized fluorescence photobleaching recovery (PFPR) has been applied for the first time to investigation of the rotational correlation time of the myosin head in muscle fibers. This is a novel application of PFPR because it is the first time PFPR has been applied to a sample which is not cylindrically symmetric about the optical axis. Therefore we present a method for analysis of PFPR results from an oriented sample such as the muscle fibers aligned perpendicularly to the optical axis used here. Control experiments performed on fluorescently labeled myosin heads in solution demonstrate that, under some conditions, our PFPR apparatus can easily measure a rotational correlation time of less than 200 μs. Validity of this application of PFPR to muscle fibers is provided by the agreement of our results with published results from a variety of other spectroscopic techniques. In particular, using glycerinated rabbit psoas muscle fibers, we find that for relaxed fibers and isometrically contracting fibers, the myosin heads undergo high-amplitude rotations on the submillisecond time domain. For fibers in rigor the myosin heads are highly oriented and nearly immobile. For fibers in ADP the myosin heads are highly ordered in a distribution quite different from that in rigor, and they are slightly more mobile than in rigor.