Metalloproteins and protein-protein complexes investigated by CW and pulsed EPR spectroscopy

One of the central research topics in the field of biophysical chemistry is the structure and function of membrane proteins involved in energy transduction. Both, the aerobic and the anaerobic respiration include electron transfer and proton translocation across the mitochondrial and bacterial membranes. These electron transfer processes lead to changes in oxidation states of cofactors some of which are paramagnetic. Therefore, EPR spectroscopy is the method of choice to obtain electronic and structural information directly related to the function of the respiratory chain proteins. In this work, multifrequency continuous wave (CW) and pulsed EPR spectroscopy has been used to characterize the molybdenum active site of polysulfide reductase (Psr) from the anaerobic bacterium Wolinella succinogenes and the protein-protein complex between cytochrome c oxidase (CcO) and cytochrome c from the aerobic bacterium Paracoccus denitrificans. Molybdenum in Psr-Psr is an enzyme essential for the sulfur respiration of Wolinella succinogenes. Biochemical studies suggested that the active site of this enzyme contains a mononuclear Mo center, which catalyzes the reduction of the substrate polysulfide to sulfide. Until now there is no crystal structure available for Psr. Consequently, current characterizations of this enzyme have to rely on biochemical and spectroscopic investigations. Within the present work, CW and modern pulsed EPR techniques were applied to investigate its catalytically active site. In the first part of this thesis, different redox agents have been used to generate paramagnetic states of Psr. Multifrequency CW-EPR spectroscopy was applied to identify the Mo(V) states. Using simulations of the experimental spectra, three spectroscopically distinct states have been identified based on the Mo hyperfine- and g-tensor values. Comparison of their EPR parameters with those of related enzymes indicated five or six sulfur ligands at the Mo center depending on the state. The state generated by addition of polysulfide is suggested to be the catalytically active form, in which the Mo is coordinated by a sulfur of the polysulfide chain as the sixth ligand. 33S (I = 3/2) labeled polysulfide was prepared to probe the proximity of the polysulfide to the molybdenum center via its hyperfine coupling. 1D-ESEEM and 2D122 HYSCORE spectroscopy was used to detect these hyperfine and quadrupole interactions, which are too small to be observed in conventional CW EPR spectra. To date there has been only one pulsed-EPR study involving a 33S nucleus [Finazzo et.al. 2003]. The reasons are that this nucleus has a high nuclear spin of I = 3/2 and a large nuclear quadrupole moment in addition to the low Larmor frequency. All these make the detection of sulfur and the extraction of structural information demanding. However, analysis of the 2D-data led to a Mo(V) 33S distance in a range of about 2 to 2.5 Å. Mo-S distances found in molybdenum enzymes of the same family are in a range of 1.8 to 2.8 Å suggesting that the 33S is indeed the sixth ligand of the Mo(V) center and demonstrating that polysulfide is the actual substrate for this enzyme. Thus HYSCORE experiments have been proved to be a powerful technique to gain further insight into the active site structures of molybdenum enzymes and the trafficking of substrate atoms during catalysis. Density functional theory (DFT) calculations together with quantitative numerical simulations of the 2D-data will help to obtain more structural details about the molybdenum binding site in Psr. CcO:cytochrome c complex Protein-protein complex formation is an important step in energy conversion biological processes such as respiration and photosynthesis. These protein-protein complexes are involved in long range electron transfer reactions and are known to be of transient nature. Within the bacterial and mitochondrial respiratory electron transport chains such a complex is formed between CcO and cytochrome c. Upon complex formation cytochrome c donates the electrons required for the CcO catalyzed reduction of dioxygen to water. Here, the protein-protein complex formation between CcO and cytochrome c from Paracoccus denitrificans was investigated by pulsed EPR spectroscopy. The idea was to use the relaxation enhancement due to the distance and orientation dependent magnetic dipole-dipole interaction between the paramagnetic centers in the different CcO constructs and cytochromes. Two-pulse electron spin echo experiments were carried out on mixtures of the CuA containing soluble subunit II or the full size CcO with the physiological partner cytochrome c552 or horse heart cytochrome c. Significantly enhanced relaxation of CuA due to specific protein-protein complex formation has been observed in all four cases. In contrast the non-binding cytochrome c1 showed only a very weak relaxation enhancement due to unspecific protein-protein interactions. The echo decays of the slowly relaxing observer spin (CuA of CcO) measured in the absence and presence of the fast relaxing spin (Fe(III) of cytochrome c) permitted the extraction of the pure dipolar relaxation contributions for the different complexes. Measurements at different temperatures proved the dipolar nature of the relaxation enhancement. Furthermore, it was demonstrated experimentally that this approach also works for the full-size CcO, which contains four paramagnetic metal centers, in complex with cytochrome c. Quantitative simulations of the data suggest a broad distribution in distances (2 - 4 nm) and orientations between the CuA and Fe(III) in the complex between CcO and cytochrome c. High-field EPR spectroscopy will be useful to further analyze and prove these complex structures. Within the present work, it has been shown that pulsed relaxation enhancement experiments can be used to investigate the distance and relative orientation between paramagnetic metal centers. Furthermore, it has been demonstrated on a qualitative level, that this method can be used complimentary to other biophysical approaches to study transient electron transfer protein-protein complexes. Finally, within this work it has been proven that this method can be applied also to biological systems where more than two paramagnetic centers are present. This is particularly interesting for supercomplexes between membrane proteins.Eines der zentralen Forschungsziele in der biophysikalischen Chemie ist die Aufklärung der Struktur und Funktion von Membranproteinen, die in Energieübertragungswegen eine Rolle spielen. Sowohl die aerobe als auch die anaerobe Atmung beinhalten Elektronentransfer und Protonentranslokation durch die mitochondrialen und bakteriellen Membranen. Die Elektronentransferprozesse führen zu Änderungen im Oxidationszustand der beteiligten Kofaktoren, wodurch paramagnetische Spezies entstehen können. Aus diesem Grund ist die elektronenparamagnetische Resonanzspektroskopie (EPR-Spektroskopie) die Methode der Wahl, um Informationen über die elektronische und molekulare Struktur der paramagnetischen Intermediate zu erhalten. Diese strukturellen Informationen sind wiederum direkt verknüpft mit Erkenntnissen über die Funktion der Proteine der Atmungskette. In dieser Arbeit wurden Multifrequenz-Continuous-Wave- (CW) und Puls-EPR-Spektroskopie verwendet, um die Molybdänbindungsstelle in Polysulfidreduktase (Psr) aus dem anaeroben Bakterium Wolinella succinogenes sowie den Protein-Protein-Komplex zwischen Cytochrom c-Oxidase (CcO) und Cytochrom c aus dem aeroben Bakterium Paracoccus denitrificans zu charakterisieren. Molybdän in Psr-Psr ist ein essentielles Enzym für die Schwefelatmung von Wolinella succinogenes. Biochemische Studien deuten darauf hin, dass das aktive Zentrum dieses Enzyms ein mononukleares Molybdänzentrum enthält, das die Reduktion des Polysulfidsubstrats zu Sulfid katalysiert. Da keine Kristallstruktur von Psr existiert, müssen biochemische und spektroskopische Methoden angewandt werden, um strukturelle Informationen über dieses Enzym zu gewinnen. In der vorliegenden Arbeit wurden daher CW- und moderne Puls-EPR-Techniken verwendet, um das katalytisch aktive Zentrum zu untersuchen. Im ersten Teil dieser Dissertation wurden verschiedene Reduktionsmittel eingesetzt, um paramagnetische Zustände von Psr zu generieren. Anschließend wurde Multifrequenz-EPR-Spektroskopie zur Identifikation der Mo(V)-Spezies verwendet. Mit Hilfe von Simulationen der experimentellen Spektren konnten – basierend auf Molybdän-Hyperfeinkopplungs- und g-Tensorwerten – drei verschiedene Zustände identifiziert werden. Auf Grund eines Vergleichs dieser EPR-Parameter mit denen verwandter und gut charakterisierter Enzyme konnte gefolgert werden, dass je nach Zustand fünf oder sechs Schwefelliganden an das Molybdänzentrum koordiniert sind. Derjenige Zustand, der durch Zugabe von Polysulfid entsteht, wurde als katalytisch aktive Form vorgeschlagen. In diesem aktiven Zustand sollte als sechster Ligand ein Schwefelatom der Polysulfidkette an das Molybdän gebunden sein. Um die postulierte Nähe des Polysulfids zum Molybdänzentrum über Hyperfeinwechselwirkungen nachzuweisen, wurde 33S-markiertes Polysulfid synthetisiert und als Substrat eingesetzt. Im Anschluss daran wurden 1D-ESEEM- und 2D-HYSCORE-Spektroskopie zur Detektion der 33SHyperfein- und 33S-Quadrupolwechselwirkungen verwendet, da diese Wechselwirkungen zu klein waren, um in konventionellen CW-EPR-Spektren beobachtet werden zu können. Bis heute existiert nur eine einzige Puls-EPR-Studie, die 33S-Wechselwirkungen behandelt, da der 33S-Kern einen Kernspin I = 3/2, ein großes Kernquadrupolmoment und eine niedrige Larmorfrequenz aufweist. All diese Eigenschaften machen es schwer, 33S zu detektieren und aus den 33S-Spektren strukturelle Informationen zu gewinnen. In dieser Arbeit war es jedoch mittels Analyse der 2D-Daten möglich, den Mo(V)-33S-Abstand auf einen Bereich von 2 bis 2.5 Å einzugrenzen. Bekannte Mo-S-Abstände für Molybdänenzyme der gleichen Familie liegen zwischen 1.8 und 2.8 Å, was den Schluss nahe legt, dass 33S aus Polysulfid tatsächlich der sechste Ligand des Mo(V)-Zentrums. Damit konnte demonstriert werden, dass Polysulfid das Substrat von Psr ist. Die präsentierte Untersuchung unterstreicht, dass HYSCORE-Experimente eine wirkungsvolle Methode darstellen, um detaillierte Einblicke in die Strukturen von aktiven Zentren von Molybdänenzymen und den Reaktionsweg von Substratatomen während der Katalyse zu gewinnen. Dichtefunktionaltheorie-Rechnungen (DFT-Rechnungen) und quantitative numerische Simulationen der 2D-Daten können in diesem Zusammenhang helfen, weitere strukturelle Details über die Molybdänbindungsstelle in Psr aus den experimentellen Daten zu extrahieren. CcO:Cytochrom c-Komplex Die Bildung von Protein-Protein-Komplexen ist ein wichtiger Schritt in biologischen Prozessen der Energieumwandlung wie z.B. der Atmung oder der Photosynthese. Solche relativ kurzlebigen Protein-Protein-Komplexe sind an langreichweitigen Elektronentransferreaktionen beteiligt. In den Elektronentransportketten der bakteriellen und mitochondrialen Atmung findet die Bildung eines Komplexes zwischen CcO und Cytochrom c statt. Nach der Komplexbildung werden die Elektronen, die CcO zur Reduktion von von O2 zu H2O benötigt, von Cytochrom c auf CcO übertragen. In dieser Arbeit wurde die Komplexbildung zwischen CcO und Cytochrom c aus Paracoccus Idenitrificans mit Hilfe gepulster EPR-Spektroskopie untersucht. Hierbei sollte der Einfluss der abstands- und orientierungsabhängigen Elektronenspin-Dipol-Dipol- Wechselwirkung zwischen verschiedenen paramagnetischen Zentren auf die Elektronenspin-Relaxation (‚relaxation enhancement’) ausgenutzt werden, um verschiedene CcO-Cytochrom-Komplexe zu charakterisieren. Daher wurden Zweipuls-Elektronenspinecho-Experimente auf Mischungen aus der löslichen Untereinheit II von CcO, die das CuA-Zentrum enthält, bzw. der kompletten CcO mit Cytochrom c552, dem physiologischen Partner, oder Cytochrom c aus Pferdeherzen angewandt. In allen vier Fällen konnte eine deutlich verstärkte Relaxation des CuA, die durch die Bildung spezifischer Protein-Protein-Komplexe verursacht wurde, beobachtet werden. Im Gegensatz dazu zeigte sich in Experimenten mit dem nichtbindenden Cytochrom c1 nur eine sehr geringe Verstärkung der Relaxation auf Grund von unspezifischen Protein-Protein-Wechselwirkungen. Ein Vergleich der Echozerfälle des langsam relaxierenden Beobachter-Spins (CuA in CcO) in An- bzw. Abwesenheit des schnell relaxierenden Elektronenspins (Fe(III) im Cytochrom) erlaubte es, die rein dipolaren Relaxationsbeiträge für die verschiedenen Komplexe zu separieren und getrennt zu untersuchen. Durch temperaturabhängige Messungen war es möglich, den dipolaren Charakter der beobachteten Verstärkung der Relaxation zu beweisen. Schließlich wurde in dieser Arbeit gezeigt, dass die verwendete experimentelle Herangehensweise auch für den CcO-Cytochrom c-Komplex mit der kompletten CcO, die insgesamt vier paramagnetische Zentren enthält, funktioniert. Quantitative Simulationen der erhaltenen Daten für die verschiedenen Komplexe ergaben eine breite Abstands- (2-4 nm) und Orientierungsverteilung für die relative Anordnung der CuA- und Fe(III)-Zentren. Um die Strukturen der Komplexe noch detaillierter zu analysieren und die bisherigen Ergebnisse zu untermauern, werden Hochfeld-EPR-Experimente durchgeführt werden. Zusammengefasst konnte in der vorliegenden Arbeit gezeigt werden, dass der Einfluss dipolarer Relaxation auf die Ergebnisse gepulste EPR-Experimente dazu verwendet werden kann, Abstände zwischen und relative Orientierungen von paramagnetischen Metallzentren zu untersuchen. Weiterhin wurde auf einem qualitativen Niveau demonstriert, dass diese – zu anderen biophysikalischen Techniken komplementäre –Methode geeignet ist, um kurzlebige Elektronentransfer-Protein-Protein-Komplexe zu studieren. Schließlich konnte in dieser Arbeit experimentell belegt werden, dass die beschriebene Methode auf biologische Systeme mit mehr als zwei paramagnetischen Zentren angewandt werden kann. Dieses Resultat ist insbesondere von Interesse für zukünftige Charakterisierungen von Membranprotein-Superkomplexen

Multifrequency cw-EPR investigation of the catalytic molybdenum cofactor of polysulfide reductase from Wolinella succinogenes

Electron paramagnetic resonance (EPR) spectra of the molybdenum centre in polysulfide reductase (Psr) from Wolinella succinogenes with unusually high G-tensor values have been observed for the first time. Three different Mo states have been generated (by the addition of the substrate polysulfide and different redox agents) and analysed by their G- and hyperfine tensors using multifrequency (S-, X- and Q-band) cw-EPR spectroscopy. The unusually high G-tensor values are attributed to a large number of sulfur ligands. Four sulfur ligands are assumed to arise from two pterin cofactors; one additional sulfur ligand was identified from mutagenesis studies to be a cysteine residue of the protein backbone. One further sulfur ligand is proposed for two of the Mo states, based on the experimentally observed shift of the g value. This sixth sulfur ligand is postulated to belong to the polysulfide substrate consumed within the catalytic reaction cycle of the enzyme. The influence of the co-protein sulfur transferase on the Mo G-tensor supports this assignment

EPR Characterization of a Rigid Bis-TEMPO-Bis-Ketal for Dynamic Nuclear Polarization

International audienceWe have characterized the rigid binitroxide radical bis-TEMPO-bis-Ketal (bTbK) by continuous-wave (CW) and pulsed electron paramagnetic resonance (EPR) spectroscopy performed at X-band (9 GHz) and G-band (180 GHz) frequencies. bTbK has been successfully used for dynamic nuclear polarization (DNP)-enhanced solid-state nuclear magnetic resonance (SS-DNP) experiments based on the cross-effect, which involves two electrons and one nuclear spin, and gave very high signal enhancements. For a quantitative description of the polarization enhancements and their excitation frequency profile, a detailed information about the values and relative orientation of the magnetic hyperfine-, dipolar-, g-tensors and the exchange interaction of the two unpaired electron spins within the molecule is mandatory. We have determined these tensors and their relative orientation by CW-EPR spectra and pulsed electron double resonance experiments in frozen solution. The potential of using the cross-effect also for DNP in liquid solutions has been experimentally investigated by room-temperature high-field DNP experiments performed at 9.2 T

Determination of the Proton Environment of High Stability Menasemiquinone Intermediate in Escherichia coli Nitrate Reductase A by Pulsed EPR

International audienceBackground: Escherichia coli nitrate reductase A highly stabilizes a semiquinone catalytic intermediate. Results: Three proton hyperfine couplings to this radical with atypical characteristics are characterized. Conclusion: Semiquinone binding is strongly asymmetric and occurs via a single short in-plane H-bond. Significance: Learning how the protein environment tunes the semiquinone properties is crucial for understanding the quinol utilization mechanism by energy-transducing enzymes

Direct evidence for nitrogen ligation to the high stability semiquinone intermediate in Escherichia coli nitrate reductase A

The membrane-bound heterotrimeric nitrate reductase A (NarGHI) catalyzes the oxidation of quinols in the cytoplasmic membrane of Escherichia coli and reduces nitrate to nitrite in the cytoplasm. The enzyme strongly stabilizes a menasemiquinone intermediate at a quinol oxidation site (Q(D)) located in the vicinity of the distal heme b(D). Here molecular details of the interaction between the semiquinone radical and the protein environment have been provided using advanced multifrequency pulsed EPR methods. (14)N and (15)N ESEEM and HYSCORE measurements carried out at X-band ( approximately 9.7 GHz) on the wild-type enzyme or the enzyme uniformly labeled with (15)N nuclei reveal an interaction between the semiquinone and a single nitrogen nucleus. The isotropic hyperfine coupling constant A(iso)((14)N) approximately 0.8 MHz shows that it occurs via an H-bond to one of the quinone carbonyl group. Using (14)N ESEEM and HYSCORE spectroscopies at a lower frequency (S-band, approximately 3.4 GHz), the (14)N nuclear quadrupolar parameters of the interacting nitrogen nucleus (kappa = 0.49, eta = 0.50) were determined and correspond to those of a histidine N(delta), assigned to the heme b(D) ligand His-66 residue. Moreover S-band (15)N ESEEM spectra enabled us to directly measure the anisotropic part of the nitrogen hyperfine interaction (T((15)N) = 0.16 MHz). A distance of approximately 2.2 Abetween the carbonyl oxygen and the nitrogen could then be calculated. Mechanistic implications of these results are discussed in the context of the peculiar properties of the menasemiquinone intermediate stabilized at the Q(D) site of NarGHI

Optimization of Transversal Relaxation of Nitroxides for Pulsed Electron-Electron Double Resonance Spectroscopy in Phospholipid Membranes

Pulsed electron-electron double resonance (PELDOR) spectroscopy is increasingly applied to spin-labeled membrane proteins. However, after reconstitution into liposomes, spin labels often exhibit a much faster transversal relaxation (T-m) than in detergent micelles, thus limiting application of the method in lipid bilayers. In this study, the main reasons for enhanced transversal relaxation in phospholipid membranes were investigated systematically by use of spin-labeled derivatives of stearic acid and phosphatidylcholine as well as spin-labeled derivatives of the channel-forming peptide gramicidin A under the conditions typically employed for PELDOR distance measurements. Our results clearly show that dephasing due to instantaneous diffusion that depends on dipolar interaction among electron spins is an important contributor to the fast echo decay in cases of high local concentrations of spin labels in membranes. The main difference between spin labels in detergent micelles and membranes is their local concentration. Consequently, avoiding spin clustering and suppressing instantaneous diffusion is the key step for maximizing PELDOR sensitivity in lipid membranes. Even though proton spin diffusion is an important relaxation mechanism, only in samples of low local concentrations does deuteration of acyl chains and buffer significantly prolong T-m. In these cases, values of up to 7 mu s have been achieved. Furthermore, our study revealed that membrane composition and labeling position in the membrane can also affect T-m, either by promoting the segregation of spin-labeled species or by altering their exposure to matrix protons. Effects of other experimental parameters including temperature (&lt; 50 K), presence of oxygen, and cryoprotectant type are negligible under our experimental conditions.</p

Determination of the proton environment of high stability Menasemiquinone intermediate in Escherichia coli nitrate reductase A by pulsed EPR

Escherichia coli nitrate reductase A (NarGHI) is a membrane-bound enzyme that couples quinol oxidation at a periplasmically oriented Q-site (Q(D)) to proton release into the periplasm during anaerobic respiration. To elucidate the molecular mechanism underlying such a coupling, endogenous menasemiquinone-8 intermediates stabilized at the Q(D) site (MSQ(D)) of NarGHI have been studied by high-resolution pulsed EPR methods in combination with (1)H2O/2H2O exchange experiments. One of the two non-exchangeable proton hyperfine couplings resolved in hyperfine sublevel correlation (HYSCORE) spectra of the radical displays characteristics typical from quinone methyl protons. However, its unusually small isotropic value reflects a singularly low spin density on the quinone carbon α carrying the methyl group, which is ascribed to a strong asymmetry of the MSQ(D) binding mode and consistent with single-sided hydrogen bonding to the quinone oxygen O1. Furthermore, a single exchangeable proton hyperfine coupling is resolved, both by comparing the HYSCORE spectra of the radical in 1H2O and 2H2O samples and by selective detection of the exchanged deuterons using Q-band 2H Mims electron nuclear double resonance (ENDOR) spectroscopy. Spectral analysis reveals its peculiar characteristics, i.e. a large anisotropic hyperfine coupling together with an almost zero isotropic contribution. It is assigned to a proton involved in a short ∼1.6 Å in-plane hydrogen bond between the quinone O1 oxygen and the Nδ of the His-66 residue, an axial ligand of the distal heme b(D). Structural and mechanistic implications of these results for the electron-coupled proton translocation mechanism at the Q(D) site are discussed, in light of the unusually high thermodynamic stability of MSQ(D)