Kinetics of Proton Coupled Electron Transfer Reactions in Enzyme Complexes of the Respiratory Chain

Protonen gekoppelter Elektronentransfer ist ein zentraler Bestandteil der chemiosmotischen Theorie. Die Beschreibung seiner Natur ist aufgrund seines transienten Charakters eine komplexe Aufgabe. Elektrometrische Messungen stellen hier eine große Hilfe in der Erfassung von Ladungsverschiebungen dar. Sie erlauben die zeitliche Beschreibung des Elektronen- und Protonentransportes, der mit kaum einer geeigneten Methode beobachtet werden kann, und geben Einblick in deren molekularen Mechanismus. Diese Technik wurde hier an drei verschiedenen Membrankomplexen der Atmungskette angewandt: der Cytochrom c Oxidase (COX) aus Paracoccus denitrificans, der Quinol-Fumarat-Reduktase (QFR) aus Wolinella succinogenes und dem bc(1)-Komplex aus Saccharomyces cerevisiae. Hinsichtlich der experimentellen Vorgehensweise für kinetische Untersuchungen stellte sich ein übergreifendes Problem. Neben einer schnellen Aktivierung der Enzymsysteme bedurfte es eines definierten Ausgangszustands. Ziel dieser Arbeit war das Etablieren von Bedingungen, die elektrometrische Messungen am bc(1) und der QFR erlauben, sowie die Fortführung dieser Methodik am bereits vorhandenen System der COX. Cytochrom c Oxidase Elektrometrische Untersuchungen an der COX wurden basierend auf Vorarbeiten weitergeführt. Insbesondere die Ladungsverschiebungen nach Photoreduktion ausgehend vom völlig oxidierten Zustand rückten in den Fokus. In diesem Schritt wird ein Proton aufgenommen, während Häm a vom angeregten Zustand eines Rutheniumkomplexes reduziert wird. Dieses Verhalten ist unabhängig vom heterogenen Ausgangszustand der COX, er wird jedoch durch eine Änderung des pH-Wertes beeinflußt. Der heterogene Ausgangszustand im O-E-Übergang wurde in einem sequentiellen Modell diskutiert. Dabei wurde auf die Diskrepanz zwischen den elektrometrischen und den publizierten spektroskopischen Messungen hingewiesen. Während spektroskopisch die Cu(A)-Oxidation und Häm a-Reduktion in einer Phase verliefen, wurde in den elektrometrischen Messungen eine zweite deutlich langsamere Phase für den Protonentransfer beobachtet. Ein sequentielles Reaktionsmodell führte hier zu einem Widerspruch. Die Auswirkungen auf die Natur der Kopplung von Häm a und dem aufgenommenen Proton wurden diskutiert. Die Kinetik der Ladungsverschiebung wurde detailliert anhand der Temperaturabhängigkeit und des Isotopeneffektes untersucht und mit den Ergebnissen aus den Messungen an einem thermophilen Enzym, der ba(3)-Oxidase aus Thermus thermophilus, verglichen. Quinol-Fumarat-Reduktase Für eine schnelle Aktivierung der QFR wurde ein caged Fumarat synthetisiert, das nach Photolyse zu einer schnellen Erhöhung der Fumaratkonzentration führte. Die neue Substanz wurde bezüglich einer möglichen Verwendung für die QFR charakterisiert. Die Freisetzung erfolgte mit einer Zeitkonstante von 0,1 ms und aktivierte die QFR nur im photolysierten Zustand. Aufgrund der Photochemie der metallischen Kofaktoren in der QFR konnten jedoch keine kinetischen Messungen durchgeführt werden. Da die photochemisch induzierten Elektronenbewegungen in der QFR mit zunehmender Wellenlänge abnahmen, wurde eine neue Substanz vorgeschlagen, die im sichtbaren Spektralbereich gespalten werden kann. bc(1)-Komplex Der bc(1)-Komplex kann wie die COX durch einen Rutheniumkomplex aktiviert werden. Ein dimerer sowie ein an Cytochrom c gekoppelter Rutheniumkomplex wurde hierfür anhand von Literaturdaten synthetisiert, und die Verbindungen wurden hinsichtlich ihrer Eigenschaften nach Lichtanregung charakterisiert. Für die elektrometrischen Messungen wurde der bc(1)-Komplex in Proteoliposomen rekonstituiert, und der Ausgangszustand des bc(1)-Komplexes unter reduktiven und oxidativen Bedingungen eingestellt. Mit dem dimeren Rutheniumkomplex wurden elektrometrische Experimente durchgeführt, die zu zwei Phasen in der Spannungsantwort führten. Die Daten wurden zusammen mit den publizierten spektroskopischen diskutiert. Dabei wurde eine schnelle elektrogene Phase einem Elektronentransfers zwischen Häm c(1) und dem Eisen-Schwefel-Cluster des Rieske-Proteins zugeordnet. Eine langsamere Phase erwies sich sensitiv gegenüber Antimycin und spiegelt Vorgänge unter der Kontrolle der Q(i)-Bindungsstelle wider.Proton coupled electron transfer is an essential part of the chemi-osmotic theory. Its description poses a complex experimental challenge due to its transient character. Electrometric measurements are a great help in the acquisition of charge translocation processes. They allow to follow in a time-resolved manner the transport of electrons and protons, respectively. In particular the latter are difficult to detect with other techniques. Potential measurements at a black lipid membrane have been performed for three different enzyme complexes from the respiratory chain: the cytochrome c oxidase from Paracoccus denitrificans (COX), the quinol:fumarate reductase from Wolinella succinogenes (QFR) and the bc(1)-complex from Saccharomyces cerevisiae. There are common problems with regard of the experimental strategy for kinetic investigations: the need for a fast and efficient activation of a particular enzyme system starting from a controlled defined state. Therefore, the goal of this work was to establish experimental conditions for electrometric measurements on the bc(1)-complex and the QFR and the continuation on the already implemented system for the COX. Cytochrome c oxidase The electrometric measurements on the COX were pursued based on previous achievements. In particular the charge translocating steps after photoreduction of the fully oxidized state of the enzyme were in the focus of this work. Here, a proton is taken up after heme a is going to be reduced from the excited state of an organometallic ruthenium complex. This behaviour is independent from the heterogeneous starting state. However, it may be influenced by a change of the pH-value. The consequences of a heterogeneous starting state in the O-E transition is discussed in the framework of a sequential reaction model revealing a discrepancy between the electrometric and the spectroscopic data. While the oxidation of Cu(A) proceeds spectroscopically with the reduction of heme a in a single observable phase, a second much slower phase is observed for the proton uptake in the electrometric measurements. This leads to a conflict with a sequential reaction model. The implications of this result are discussed with respect to the nature of the coupling between electron and proton transfer. The kinetics of charge translocation processes have been examined in detail by means of the temperature dependence and the isotope effect for the electron and proton transfer in the O-E transition. The results are compared with the ones for a thermophilic enzyme, i.e. the ba(3)-oxidase from Thermus thermophilus. Quinol:fumarate reductase A caged fumarate has been synthesized with the aim of a fast activation of the QFR. Photolysis of this compound leads to a fast concentration jump of fumarate. The new compound has been characterized to explore the its suitability in measurements with the QFR. The release of fumarate takes place with a time constant of 0,1 ms and it starts the enzymatic reaction of the QFR only after photolysis. However, it was not possible to perform kinetic measurements due to the photochemistry of the metallic cofactors in the QFR. Because the light-induced redox reactions in the QFR are decreasing with increasing wavelengths, the synthesis of a new compound has been proposed that may be photolyzed in the visible spectral range. bc(1)-complex The bc(1)-complex as well can be activated by a ruthenium complex as the COX. Therefore, a dimeric complex and one coupled to cytochrome c were synthesized according to literature data. The compounds were characterized, in particular after light excitation. The bc(1)-complex was reconstituted in proteoliposomes for the electrometric measurements and the starting state was prepared under oxidizing or reducing experimental conditions. Usage of the dimeric ruthenium complex in the electrometric measurements leads to two distinct phases in the photopotential over the membrane. The results were discussed and compared to published data from spectroscopic experiments. A fast electrogenic phase was assigned to electron transfer between heme c(1) and the iron-sulfur cluster of the Rieske protein. A second slower one showed a sensitivity towards antimycin and reflects processes in the Q(i) binding site

Atomistic Insight into the Role of Threonine 127 in the Functional Mechanism of Channelrhodopsin-2

Channelrhodopsins (ChRs) belong to the unique class of light-gated ion channels. The structure of channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) has been resolved, but the mechanistic link between light-induced isomerization of the chromophore retinal and channel gating remains elusive. Replacements of residues C128 and D156 (DC gate) resulted in drastic effects in channel closure. T127 is localized close to the retinal Schiff base and links the DC gate to the Schiff base. The homologous residue in bacteriorhodopsin (T89) has been shown to be crucial for the visible absorption maximum and dark–light adaptation, suggesting an interaction with the retinylidene chromophore, but the replacement had little effect on photocycle kinetics and proton pumping activity. Here, we show that the T127A and T127S variants of CrChR2 leave the visible absorption maximum unaffected. We inferred from hybrid quantum mechanics/molecular mechanics (QM/MM) calculations and resonance Raman spectroscopy that the hydroxylic side chain of T127 is hydrogen-bonded to E123 and the latter is hydrogen-bonded to the retinal Schiff base. The C=N–H vibration of the Schiff base in the T127A variant was 1674 cm−1, the highest among all rhodopsins reported to date. We also found heterogeneity in the Schiff base ground state vibrational properties due to different rotamer conformations of E123. The photoreaction of T127A is characterized by a long-lived P2380 state during which the Schiff base is deprotonated. The conservative replacement of T127S hardly affected the photocycle kinetics. Thus, we inferred that the hydroxyl group at position 127 is part of the proton transfer pathway from D156 to the Schiff base during rise of the P3530 intermediate. This finding provides molecular reasons for the evolutionary conservation of the chemically homologous residues threonine, serine, and cysteine at this position in all channelrhodopsins known so far

Temporal evolution of helix hydration in a light-gated ion channel correlates with ion conductance

The discovery of channelrhodopsins introduced a new class of light-gated ion channels, which when genetically encoded in host cells resulted in the development of optogenetics. Channelrhodopsin-2 from Chlamydomonas reinhardtii, CrChR2, is the most widely used optogenetic tool in neuroscience. To explore the connection between the gating mechanism and the influx and efflux of water molecules in CrChR2, we have integrated light-induced time- resolved infrared spectroscopy and electrophysiology. Cross-correlation analysis revealed that ion conductance tallies with peptide backbone amide I vibrational changes at 1,665(−) and 1,648(+) cm−1. These two bands report on the hydration of transmembrane α-helices as concluded from vibrational coupling experiments. Lifetime distribution analysis shows that water influx proceeded in two temporally separated steps with time constants of 10 μs (30%) and 200 μs (70%), the latter phase concurrent with the start of ion conductance. Water efflux and the cessation of the ion conductance are synchronized as well, with a time constant of 10 ms. The temporal correlation between ion conductance and hydration of helices holds for fast (E123T) and slow (D156E) variants of CrChR2, strengthening its functional significance

Light-induced helix movements in channelrhodopsin-2

Channelrhodopsin-2 (ChR2) is a cation-selective light-gated channel from Chlamydomonas reinhardtii (Nagel G, Szellas T, Huhn W, Kateriya S, Adeishvili N, Berthold P, et al. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc Natl Acad Sci USA 2003;100:13940-5), which has become a powerful tool in optogenetics. Two-dimensional crystals of the slow photocycling C128T ChR2 mutant were exposed to 473 nm light and rapidly frozen to trap the open state. Projection difference maps at 6Å resolution show the location, extent and direction of light-induced conformational changes in ChR2 during the transition from the closed state to the ion-conducting open state. Difference peaks indicate that transmembrane helices (TMHs) TMH2, TMH6 and TMH7 reorient or rearrange during the photocycle. No major differences were found near TMH3 and TMH4 at the dimer interface. While conformational changes in TMH6 and TMH7 are known from other microbial-type rhodopsins, our results indicate that TMH2 has a key role in light-induced channel opening and closing in ChR2

The retinal structure of channelrhodopsin-2 assessed by resonance Raman spectroscopy

Nack M, Radu I, Bamann C, Bamberg E, Heberle J. The retinal structure of channelrhodopsin-2 assessed by resonance Raman spectroscopy. FEBS Letters. 2009;583(22):3676-3680.Channelrhodopsin-2 mediates phototaxis in green algae by acting as a light-gated cation channel. As a result of this property, it is used as a novel optogenetic tool in neurophysiological applications. Structural information is still scant and we present here the first resonance Raman spectra of channelrhodopsin-2. Spectra of detergent solubilized and lipid-reconstituted protein were recorded under pre-resonant conditions to exclusively probe retinal in its electronic ground state. All-trans retinal was identified to be the favoured configuration of the chromophore but significant contributions of 13-cis were detected. Pre-illumination hardly changed the isomeric composition but small amounts of presumably 9-cis retinal were found in the light-adapted state. Spectral analysis suggested that the Schiff base proton is strongly hydrogen-bonded to a nearby water molecule. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved

High-temperature solution NMR structure of TmCsp

Cold shock proteins (Csps) are assumed to play a central role in the regulation of gene expression under cold shock conditions. Acting as single-stranded nucleic acid-binding proteins, they trigger the translation process and are therefore involved in the compensation of the influence of low temperatures (cold shock) upon the cell metabolism. However, it is unknown so far how Csps are switched on and off as a function of temperature. The aim of the present study is the study of possible structural changes responsible for this switching process. 1H-15N HSQC spectra recorded at different temperatures and chemical-shift analysis have indicated subtle conformational changes for the cold-shock protein from the hyperthermophilic bacterium Thermotoga maritima (TmCsp) when the temperature is elevated from 303 K to its physiological temperature (343 K). The three-dimensional structure of TmCsp was determined by nuclear magnetic resonance (NMR) spectroscopy at 343 K to obtain quantitative information concerning these structural changes. By use of residual dipolar couplings, the loss of NOE information at high temperature could be compensated successfully. Most pronounced conformational changes compared with room-temperature conditions are observed for amino acid residues closely neighbored to two characteristic β-bulges and a well-defined loop region of the protein. Because the residues shown to be responsible for the interaction of TmCsp with single-stranded nucleic acids can almost exclusively be found within these regions, nucleic acid-binding activity might be down-regulated with increasing temperature by the described conformational changes

Channelrhodopsin-2 is a leaky proton pump

Since its discovery, the light-gated cation channel Channelrhodopsin-2 (ChR2) has proven to be a long-sought tool for the noninvasive, light-activated control of neural cells in culture and in living animals. Although ChR2 is widely used in neurobiological applications, little is known about its molecular mechanism. In this work, the unitary conductance of ChR2 was determined for different cations, for example 40 fS at 200 mM NaCl and −60 mV, using noise analysis. The kinetics of the ion channel obtained by noise analysis is in excellent agreement with the photocurrent kinetics obtained by voltage-clamp and time-resolved spectroscopy. The inward rectification of the channel could be explained by the single channel parameters. ChR2 represents an ion channel with a 7 transmembrane helix motif, even though the sequence homology of its essential amino acids to those of the light-driven H+ pump bacteriorhodopsin (bR) is high. Here, we also show that when ChR2 is expressed in electrofused giant HEK293 cells or reconstituted on planar lipid membranes, it can indeed act as an outwardly driven H+ pump, demonstrating that ChR2 is bifunctional, and in-line with other microbial rhodopsins, a H+ pump but with a leak that shows ion channel properties