The Domon Family of Plant Plasma Membrane B-Type Cytochromes: Heterologous Expression, Biochemical Characterization and Physiological Roles in Vivo

The DOMON domain is a domain widespread in nature, predicted to fold in a β-sandwich structure. In plants, AIR12 is constituted by a single DOMON domain located in the apoplastic space and is GPI-modified for anchoring to the plasma membrane. Arabidopsis thaliana AIR12 has been heterologously expressed as a recombinant protein (recAtAIR12) in Pichia pastoris. Spectrophotometrical analysis of the purified protein showed that recAtAir12 is a cytochrome b. RecAtAIR12 is highly glycosylated, it is reduced by ascorbate, superoxide and naftoquinones, oxidised by monodehydroascorbate and oxygen and insensitive to hydrogen peroxide. The addition of recAtAIR12 to permeabilized plasma membranes containing NADH, FeEDTA and menadione, caused a statistically significant increase in hydroxyl radicals as detected by electron paramagnetic resonance. In these conditions, recAtAIR12 has thus a pro-oxidant role. Interestingly, AIR12 is related to the cytochrome domain of cellobiose dehydrogenase which is involved in lignin degradation, possibly via reactive oxygen species (ROS) production. In Arabidopsis the Air12 promoter is specifically activated at sites where cell separations occur and ROS, including •OH, are involved in cell wall modifications. air12 knock-out plants infected with Botrytis cinerea are more resistant than wild-type and air12 complemented plants. Also during B. cinerea infection, cell wall modifications and ROS are involved. Our results thus suggest that AIR12 could be involved in cell wall modifying reactions by interacting with ROS and ascorbate. CyDOMs are plasma membrane redox proteins of plants that are predicted to contain an apoplastic DOMON fused with a transmembrane cytochrome b561 domain. CyDOMs have never been purified nor characterised. The trans-membrane portion of a soybean CyDOM was expressed in E. coli but purification could not be achieved. The DOMON domain was expressed in P. pastoris and shown to be itself a cytochrome b that could be reduced by ascorbate.

Zur Struktur und Funktion der Typ-3-Kupferproteine sowie der Lichtsammlerkomplexe LHCI und LHCII

In der vorliegenden Arbeit wurden die bioinformatischen Methoden der Homologie-Modellierung und Molekularen Modellierung dazu benutzt, die dreidimensionalen Strukturen verschiedenster Proteine vorherzusagen und zu analysieren. Experimentelle Befunde aus Laborversuchen wurden dazu benutzt, die Genauigkeit der Homologie-Modelle zu erhöhen. Die Ergebnisse aus den Modellierungen wurden wiederum dazu benutzt, um neue experimentelle Versuche vorzuschlagen. Anhand der erstellten Modelle und bekannten Kristallstrukturen aus der Protein-Datenbank PDB wurde die Struktur-Funktionsbeziehung verschiedener Tyrosinasen untersucht. Dazu gehörten sowohl die Tyrosinase des Bakteriums Streptomyces als auch die Tyrosinase der Hausmaus. Aus den vergleichenden Strukturanalysen der Tyrosinasen resultierten Mechanismen für die Monophenolhydroxylase-Aktivität der Tyrosinasen sowie für den Import der Kupferionen ins aktive Zentrum. Es konnte der Beweis geführt werden, daß die Blockade des CuA-Zentrums tatsächlich der Grund für die unterschiedliche Aktivität von Tyrosinasen und Catecholoxidasen ist. Zum ersten Mal konnte mit der Maus-Tyrosinase ein vollständiges Strukturmodell einer Säugetier-Tyrosinase erstellt werden, das dazu in der Lage ist, die Mechanismen bekannter Albino-Mutationen auf molekularer Ebene zu erklären. Die auf der Basis des ermittelten 3D-Modells gewonnenen Erkenntnisse über die Wichtigkeit bestimmter Aminosäuren für die Funktion wurde durch gerichtete Mutagenese an der rekombinant hergestellten Maus-Tyrosinase getestet und bestätigt. Weiterhin wurde die Struktur der Tyrosinase des Krebses Palinurus elephas durch eine niedrigaufgelöste 3D-Rekonstruktion aus elektronenmikroskopischen Bildern aufgeklärt. Der zweite große Themenkomplex umfasst die Strukturanalyse der Lichtsammlerkomplexe LHCI-730 und LHCII. Im Falle des LHCII konnte der Oligomerisierungszustand der LHCMoleküle mit diskreten Konformationen des N-Terminus korreliert werden. Auch hier kam eine Kombination von Homologie-Modellierung und einer experimentellen Methode, der Elektronen-Spin-Resonanz-Messung, zum Einsatz. Die Änderung des Oligomerisierungszustands des LHCII kontrolliert den Energiezufluß zu den Photosystemen PS I und PS II. Des Weiteren wurde ein vollständiges Modell des LHCI-730 erstellt, um die Auswirkungen gerichteter Mutagenese auf das Dimerisierungsverhalten zu untersuchen. Auf Basis dieses Modells wurden die Wechselwirkungen zwischen den Monomeren Lhca1 und Lhca4 evaluiert und potentielle Bindungspartner identifiziert.

Modification of the magnetic properties of a heterometallic wheel by inclusion of a Jahn-Teller distorted Cu(II) ion

An investigation into the physical consequences of including a Jahn-Teller distorted Cu(II) ion within an antiferromagnetically coupled ring, [R(2)NH(2)][Cr(7)CuF(8)((O(2)C(t)Bu)(16))] is reported. Inelastic neutron scattering (INS) and electron paramagnetic resonance (EPR) spectroscopic data are simulated using a microscopic spin Hamiltonian, and show that the two Cr-Cu exchange interactions must be inequivalent. One Cr-Cu exchange is found to be antiferromagnetic and the other ferromagnetic. The geometry of the Jahn-Teller elongation is deduced from these results, and shows that a Jahn-Teller elongation axis must lie in the plane of the Cr(7)Cu wheel; the elongation is not observed by X-ray crystallography, due to positional disorder of the Cu site within the wheel. An electronic structure calculation confirms the structural distortion of the Cu site.

Different dark conformations function in color-sensitive photosignaling by the sensory rhodopsin I-HtrI complex.

The haloarchaeal phototaxis receptor sensory rhodopsin I (SRI) in complex with its transducer HtrI delivers an attractant signal from excitation with an orange photon and a repellent signal from a second near-UV photon excitation. Using a proteoliposome system with purified SRI in complex with its transducer HtrI, we identified by site-directed fluorescence labeling a site (Ser(155)) on SRI that is conformationally active in signal relay to HtrI. Using site-directed spin labeling of Ser(155)Cys with a nitroxide side chain, we detected a change in conformation following one-photon excitation such that the spin probe exhibits a splitting of the outer hyperfine extrema (2A'(zz)) significantly smaller than that of the electron paramagnetic resonance spectrum in the dark state. The dark conformations of five mutant complexes that do not discriminate between orange and near-UV excitation show shifts to lower or higher 2A'(zz) values correlated with the alterations in their motility behavior to one- and two-photon stimuli. These data are interpreted in terms of a model in which the dark complex is populated by two conformers in the wild type, one that inhibits the CheA kinase (A) and the other that activates it (R), shifted in the dark by mutations and shifted in the wild-type SRI-HtrI complex in opposite directions by one-photon and two-photon reactions.

Overexpression of human copper,zinc-superoxide dismutase (SOD1) prevents postischemic injury

Superoxide and superoxide-derived oxidants have been hypothesized to be important mediators of postischemic injury. Whereas copper,zinc-superoxide dismutase, SOD1, efficiently dismutates superoxide, there has been controversy regarding whether increasing intracellular SOD1 expression would protect against or potentiate cellular injury. To determine whether increased SOD1 protects the heart from ischemia and reperfusion, studies were performed in a newly developed transgenic mouse model in which direct measurement of superoxide, contractile function, bioenergetics, and cell death could be performed. Transgenic mice with overexpression of human SOD1 were studied along with matched nontransgenic controls. Immunoblotting and immunohistology demonstrated that total SOD1 expression was increased 10-fold in hearts from transgenic mice compared with nontransgenic controls, with increased expression in both myocytes and endothelial cells. In nontransgenic hearts following 30 min of global ischemia a reperfusion-associated burst of superoxide generation was demonstrated by electron paramagnetic resonance spin trapping. However, in the transgenic hearts with overexpression of SOD1 the burst of superoxide generation was almost totally quenched, and this was accompanied by a 2-fold increase in the recovery of contractile function, a 2.2-fold decrease in infarct size, and a greatly improved recovery of high energy phosphates compared with that in nontransgenic controls. These results demonstrate that superoxide is an important mediator of postischemic injury and that increasing intracellular SOD1 dramatically protects the heart from this injury. Thus, increasing intracellular SOD1 expression may be a highly effective approach to decrease the cellular injury that occurs following reperfusion of ischemic tissues.

Structure and function in rhodopsin: Rhodopsin mutants with a neutral amino acid at E134 have a partially activated conformation in the dark state*

The Glu-134–Arg-135 residues in rhodopsin, located near the cytoplasmic end of the C helix, are involved in G protein binding, or activation, or both. Furthermore, the charge-neutralizing mutation Glu-134 to Gln-134 produces hyperactivity in the activated state and produces constitutive activity in opsin. The Glu/Asp-Arg charge pair is highly conserved in equivalent positions in other G protein-coupled receptors. To investigate the structural consequences of charge-neutralizing mutations at Glu-134 and Arg-135 in rhodopsin, single spin-labeled side chains were introduced at sites in the cytoplasmic domains of helices C (140), E (227), F (250), or G (316) to serve as “molecular sensors” of the local helix bundle conformation. In each of the spin-labeled rhodopsins, a Gln substitution was introduced at either Glu-134 or Arg-135, and the electron paramagnetic resonance spectrum of the spin label was used to monitor the structural response of the helix bundle. The results indicate that a Gln substitution at Glu-134 induces a photoactivated conformation around helices C and G even in the dark state, an observation of potential relevance to the hyperactivity and constitutive activity of the mutant. In contrast, little change is induced in helix F, which has been shown to undergo a dominant motion upon photoactivation. This result implies that the multiple helix motions accompanying photoactivation are not strongly coupled and can be induced to take place independently. Gln substitution at Arg-135 produces only minor structural changes in the dark- or light-activated conformation, suggesting that this residue is not a determinant of structure in the regions investigated, although it may be functionally important.

The Responses of Cytochrome Redox State and Energy Metabolism to Dehydration Support a Role for Cytoplasmic Viscosity in Desiccation Tolerance1

To characterize the depression of metabolism in anhydrobiotes, the redox state of cytochromes and energy metabolism were studied during dehydration of soaked cowpea (Vigna unguiculata) cotyledons and pollens of Typha latifolia and Impatiens glandulifera. Between water contents (WC) of 1.0 and 0.6 g H2O/g dry weight (g/g), viscosity as measured by electron spin resonance spectroscopy increased from 0.15 to 0.27 poise. This initial water loss was accompanied by a 50% decrease in respiration rates, whereas the adenylate energy charge remained constant at 0.8, and cytochrome c oxidase (COX) remained fully oxidized. From WC of 0.6 to 0.2 g/g, viscosity increased exponentially. The adenylate energy charge declined to 0.4 in seeds and 0.2 in pollen, whereas COX became progressively reduced. At WC of less than 0.2 g/g, COX remained fully reduced, whereas respiration ceased. When dried under N2, COX remained 63% reduced in cotyledons until WC was 0.7 g/g and was fully reduced at 0.2 g/g. During drying under pure O2, the pattern of COX reduction was similar to that of air-dried tissues, although the maximum reduction was 70% in dried tissues. Thus, at WC of less than 0.6 g/g, the reduction of COX probably originates from a decreased O2 availability as a result of the increased viscosity and impeded diffusion. We suggest that viscosity is a valuable parameter to characterize the relation between desiccation and decrease in metabolism. The implications for desiccation tolerance are discussed.

Site-directed spin labeling and chemical crosslinking demonstrate that helix V is close to helices VII and VIII in the lactose permease of Escherichia coli.

Site-directed chemical cleavage of lactose permease indicates that helix V is in close proximity to helices VII and VIII. To test this conclusion further, permease containing a biotin-acceptor domain and paired Cys residues at positions 148 (helix V) and 228 (helix VII), 148 and 226 (helix VII), or 148 and 275 (helix VIII) was affinity purified and labeled with a sulfhydryl-specific nitroxide spin label. Spin-spin interactions are observed with the 148/228 and 148/275 pairs, indicating close proximity between appropriate faces of helix V and helices VII and VIII. Little or no interaction is evident with the 148/226 pair, in all likelihood because position 226 is on the opposite face of helix VII from position 228. Broadening of the electron paramagnetic resonance spectra in the frozen state was used to estimate distance between the 148/228 and the 148/275 pairs. The nitroxides at positions 148 and 228 or 148 and 275 are within approximately 13-15 A. Finally, Cys residues at positions 148 and 228 are crosslinked by dibromobimane, a bifunctional crosslinker that is approximately 5 A. long, while no crosslinking is detected between Cys residues at positions 148 and 275 or 148 and 226. The results provide strong support for a structure in which helix V is in close proximity to both helices VII and VIII and is oriented in such a fashion that Cys-148 is closer to helix VII.

Nitric oxide synthase generates superoxide and nitric oxide in arginine-depleted cells leading to peroxynitrite-mediated cellular injury.

Besides synthesizing nitric oxide (NO), purified neuronal NO synthase (nNOS) can produce superoxide (.O2-) at lower L-Arg concentrations. By using electron paramagnetic resonance spin-trapping techniques, we monitored NO and .O2- formation in nNOS-transfected human kidney 293 cells. In control transfected cells, the Ca2+ ionophore A23187 triggered NO generation but no .O2- was seen. With cells in L-Arg-free medium, we observed .O2- formation that increased as the cytosolic L-Arg levels decreased, while NO generation declined. .O2- formation was virtually abolished by the specific NOS blocker, N-nitro-L-arginine methyl ester (L-NAME). Nitrotyrosine, a specific nitration product of peroxynitrite, accumulated in L-Arg-depleted cells but not in control cells. Activation by A23187 was cytotoxic to L-Arg-depleted, but not to control cells, with marked lactate dehydrogenase release. The cytotoxicity was largely prevented by either superoxide dismutase or L-NAME. Thus, with reduced L-Arg availability NOS elicits cytotoxicity by generating .O2- and NO that interact to form the potent oxidant peroxynitrite. Regulating arginine levels may provide a therapeutic approach to disorders involving .O2-/NO-mediated cellular injury.

Oxidation states of the manganese cluster during the flash-induced S-state cycle of the photosynthetic oxygen-evolving complex.

The Mn K-edge x-ray absorption spectra for the pure S states of the tetranuclear Mn cluster of the oxygen-evolving complex of photosystem II during flash-induced S-state cycling have been determined. The relative S-state populations in samples given 0, 1, 2, 3, 4, or 5 flashes were determined from fitting the flash-induced electron paramagnetic resonance (EPR) multiline signal oscillation pattern to the Kok model. The edge spectra of samples given 0, 1, 2, or 3 flashes were combined with EPR information to calculate the pure S-state edge spectra. The edge positions (defined as the zero-crossing of the second derivatives) are 6550.1, 6551.7, 6553.5, and 6553.8 eV for S0, S1, S2, and S3, respectively. In addition to the shift in edge position, the S0--> S1 and S1--> S2 transitions are accompanied by characteristic changes in the shape of the edge, both indicative of Mn oxidation. The edge position shifts very little (0.3 eV) for the S2--> S3 transition, and the edge shape shows only subtle changes. We conclude that probably no direct Mn oxidation is involved in this transition. The proposed Mn oxidation state assignments are as follows: S0 (II, III, IV, IV) or (III, III, III, IV), S1 (III, III, IV, IV), S2 (III, IV, IV, IV), S3 (III, IV, IV, IV).

Construction and characterization of an azurin analog for the purple copper site in cytochrome c oxidase.

A protein analog of a purple copper center has been constructed from a recombinant blue copper protein (Pseudomonas aeruginosa azurin) by replacing the loop containing the three ligands to the blue copper center with the corresponding loop of the CuA center in cytochrome c oxidase (COX) from Paracoccus denitrificans. The electronic absorption in the UV and visible region (UV-vis) and electron paramagnetic resonance (EPR) spectra of this analog are remarkably similar to those of the native CuA center in COX from Paracoccus denitrificans. The above spectra can be obtained upon addition of a mixture of Cu2+ and Cu+. Addition of Cu2+ only results in a UV-vis spectrum consisting of absorptions from both a purple copper center and a blue copper center. This spectrum can be converted to the spectrum of a pure purple copper by a prolonged incubation in the air, or by addition of excess ascorbate. The azurin mutant reported here is an example of an engineered purple copper center with the A480/A530 ratio greater than 1 and with no detectable hyperfines, similar to those of the CuA sites in COX of bovine heart and of Paracoccus denitrificans.

Distance determination in proteins using designed metal ion binding sites and site-directed spin labeling: application to the lactose permease of Escherichia coli.

As shown in the accompanying paper, the magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances in T4 lysozyme, a protein of known structure. To evaluate this potentially powerful method for general use, particularly with membrane proteins that are difficult to crystallize, both a paramagnetic metal ion binding site and a nitroxide side chain were introduced at selected positions in the lactose permease of Escherichia coli, a paradigm for polytopic membrane proteins. Thus, three individual cysteine residues were introduced into putative helix IV of a lactose permease mutant devoid of native cysteine residues containing a high-affinity divalent metal ion binding site in the form of six contiguous histidine residues in the periplasmic loop between helices III and IV. In addition, the construct contained a biotin acceptor domain in the middle cytoplasmic loop to facilitate purification. After purification and spin labeling, electron paramagnetic resonance spectra were obtained with the purified proteins in the absence and presence of Cu(II). The results demonstrate that positions 103, 111, and 121 are 8, 14, and > 23 A from the metal binding site. These data are consistent with an alpha-helical conformation of transmembrane domain IV of the permease. Application of the technique to determine helix packing in lactose permease is discussed.

Proximity of the manganese cluster of photosystem II to the redox-active tyrosine YZ.

Electron spin echo electron-nuclear double resonance (ESE-ENDOR) experiments performed on a broad radical electron paramagnetic resonance (EPR) signal observed in photosystem II particles depleted of Ca2+ indicate that this signal arises from the redox-active tyrosine YZ. The tyrosine EPR signal width is increased relative to that observed in a manganese-depleted preparation due to a magnetic interaction between the photosystem II manganese cluster and the tyrosine radical. The manganese cluster is located asymmetrically with respect to the symmetry-related tyrosines YZ and YD. The distance between the YZ tyrosine and the manganese cluster is estimated to be approximately 4.5 A. Due to this close proximity of the Mn cluster and the redox-active tyrosine YZ, we propose that this tyrosine abstracts protons from substrate water bound to the Mn cluster.

Endogenous intracellular glutathionyl radicals are generated in neuroblastoma cells under hydrogen peroxide oxidative stress.

We report the detection of endogenous intracellular glutathionyl (GS.) radicals in the intact neuroblastoma cell line NCB-20 under oxidative stress. Spin-trapping and electron paramagnetic resonance (EPR) spectroscopic methods were used for monitoring the radicals. The cells incubated with the spin trap 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) were challenged with H2O2 generated by the enzymic reaction of glucose/glucose oxidase. These cells exhibit the EPR spectrum of the GS. radical adduct of DMPO (DMPO-.SG) without exogenous reduced glutathione (GSH). The identity of this radical adduct was confirmed by observing hyperfine coupling constants identical to previously reported values in in vitro studies, which utilized known enzymic reactions, such as horseradish peroxidase and Cu/Zn superoxide dismutase, with GSH and H2O2 as substrates. The formation of the GS. radicals required viable cells and continuous biosynthesis of GSH. No significant effect on the resonance amplitude by the addition of a membrane-impermeable paramagnetic broadening agent indicated that these radicals were located inside the intact cell. N-Acetyl-L-cysteine (NAC)-treated cells produced NAC-derived free radicals (NAC.) in place of GS. radicals. The time course studies showed that DMPO-.SG formation exhibited a large increase in its concentration after a lag period, whereas DMPO-NAC. formation from NAC-treated cells did not show this sudden increase. These results were discussed in terms of the limit of antioxidant enzyme defenses in cells and the potential role of the GS. radical burst in activation of the transcription nuclear factor NF-kappa B in response to oxidative stress.

Applications of EPR with an Emphasis on Tau Fibril Structure

Substances containing unpaired electrons have been studied by electron paramagnetic resonance (EPR) for nearly 70 years. With continual development and enhancement of EPR techniques, questions have arisen regarding optimum method selection for a given sample based on its properties. In this work, radiation defects, natural lattice defects, solid organic radicals, radicals in solution, and spin-labeled proteins were analyzed using CW, pulse, and rapid scan EPR to compare methods. Studies of solid BDPA, EOe in quartz, Ns0 in diamond, and a-Si:H, showed that rapid scan could overcome many obstacles presented by other techniques, cementing rapid scan as an effective alternative to CW and pulse methods. Relaxation times of six nitroxide radicals were characterized from 0.25-34 GHz, guiding synthesis of improved nitroxides for in vivo imaging experiments. Processes contributing to T1 of DPPH in polystyrene were found through variable temperature measurements at X- and Q-band, resolving previously-reported discrepancies in relaxation properties and providing new insight into this commonly-used standard. In the history of EPR, the study of proteins is relatively new. Double electron-electron resonance (DEER) has emerged as a powerful technique for the study of amyloid fibrils, a class of protein aggregates implicated in a number of neurodegenerative disorders. Microtubule-associated protein tau forms fibrils linked to Alzheimerfs disease through seeded conversion of monomer. Self-assembly is mediated by the microtubule binding repeats in tau, and there are either three or four repeats present depending on the isoform. DEER was used to show that filaments of 3R and 4R tau are conformationally distinct and that 4R fibrils adopt a heterogeneous mixture of conformations. Populations of 4R fibril conformations, which were independently validated using a model system, can be modulated by introduction of mutations to the primary sequence or by varying fibril growth conditions. These findings provided unprecedented insights into the seed selection of tau monomers and established conformational compatibility as an important driving force in tau fibril propagation. Lastly, DEER acquisition was improved through addition of paramagnetic metal to spin-labeled protein, decreasing collection time, and through use of a novel spin label with increased T2, thereby lengthening the available acquisition window.