Electron Entry Research Articles

Functional Dissection of the Multi-Domain Di-Heme Cytochrome c550 from Thermus thermophilus

In bacteria, oxidation of sulfite to sulfate, the most common strategy for sulfite detoxification, is mainly accomplished by the molybdenum-containing sulfite:acceptor oxidoreductases (SORs). Bacterial SORs are very diverse proteins; they can exist as monomers or homodimers of their core subunit, as well as heterodimers with an additional cytochrome c subunit. We have previously described the homodimeric SOR from Thermus thermophilus HB8 (SORTTHB8), identified its physiological electron acceptor, cytochrome c 550, and demonstrated the key role of the latter in coupling sulfite oxidation to aerobic respiration. Herein, the role of this di-heme cytochrome c was further investigated. The cytochrome was shown to be composed of two conformationally independent domains, each containing one heme moiety. Each domain was separately cloned, expressed in E. coli and purified to homogeneity. Stopped-flow experiments showed that: i) the N-terminal domain is the only one accepting electrons from SORTTHB8; ii) the N- and C-terminal domains are in rapid redox equilibrium and iii) both domains are able to transfer electrons further to cytochrome c 552, the physiological substrate of the ba 3 and caa 3 terminal oxidases. These findings show that cytochrome c 550 functions as a electron shuttle, without working as an electron wire with one heme acting as the electron entry and the other as the electron exit site. Although contribution of the cytochrome c 550 C-terminal domain to T. thermophilus sulfur respiration seems to be dispensable, we suggest that di-heme composition of the cytochrome physiologically enables storage of the two electrons generated from sulfite oxidation, thereof ensuring efficient contribution of sulfite detoxification to the respiratory chain-mediated energy generation.

Alternative ground states enable pathway switching in biological electron transfer

Electron transfer is the simplest chemical reaction and constitutes the basis of a large variety of biological processes, such as photosynthesis and cellular respiration. Nature has evolved specific proteins and cofactors for these functions. The mechanisms optimizing biological electron transfer have been matter of intense debate, such as the role of the protein milieu between donor and acceptor sites. Here we propose a mechanism regulating long-range electron transfer in proteins. Specifically, we report a spectroscopic, electrochemical, and theoretical study on WT and single-mutant Cu(A) redox centers from Thermus thermophilus, which shows that thermal fluctuations may populate two alternative ground-state electronic wave functions optimized for electron entry and exit, respectively, through two different and nearly perpendicular pathways. These findings suggest a unique role for alternative or "invisible" electronic ground states in directional electron transfer. Moreover, it is shown that this energy gap and, therefore, the equilibrium between ground states can be fine-tuned by minor perturbations, suggesting alternative ways through which protein-protein interactions and membrane potential may optimize and regulate electron-proton energy transduction.

Structural insights into electron transfer in caa3-type cytochrome oxidase

Summary ParagraphCytochrome c oxidase is a member of the heme copper oxidase superfamily (HCO)1. HCOs function as the terminal enzymes in the respiratory chain of mitochondria and aerobic prokaryotes, coupling molecular oxygen reduction to transmembrane proton pumping. Integral to the enzyme’s function is the transfer of electrons from cytochrome c to the oxidase via a transient association of the two proteins. Electron entry and exit are proposed to occur from the same site on cytochrome c2–4. Here we report the crystal structure of the caa3-type cytochrome oxidase from Thermus thermophilus, which has a covalently tethered cytochrome c domain. Crystals were grown in a bicontinuous mesophase using a synthetic short-chain monoacylglycerol as the hosting lipid. From the electron density map, at 2.36 Å resolution, a novel integral membrane subunit and a native glycoglycerophospholipid embedded in the complex were identified. Contrary to previous electron transfer mechanisms observed for soluble cytochrome c, the structure reveals the architecture of the electron transfer complex for the fused cupredoxin/cytochrome c domain which implicates different sites on cytochrome c for electron entry and exit. Support for an alternative to the classical proton gate characteristic of this HCO class is presented.

Redox Properties of Lysine- and Methionine-Coordinated Hemes Ensure Downhill Electron Transfer in NrfH2A4 Nitrite Reductase

The multiheme NrfHA nitrite reductase is a menaquinol:nitrite oxidoreductase that catalyzes the 6-electron reduction of nitrite to ammonia in a reaction that involves eight protons. X-ray crystallography of the enzyme from Desulfovibrio vulgaris revealed that the biological unit, NrfH2A4, houses 28 c-type heme groups, 22 of them with low spin and 6 with pentacoordinated high spin configuration. The high spin hemes, which are the electron entry and exit points of the complex, carry a highly unusual coordination for c-type hemes, lysine and methionine as proximal ligands in NrfA and NrfH, respectively. Employing redox titrations followed by X-band EPR spectroscopy and surface-enhanced resonance Raman spectroelectrochemistry, we provide the first experimental evidence for the midpoint redox potential of the NrfH menaquinol-interacting methionine-coordinated heme (-270 ± 10 mV, z = 0.96), identified by the use of the inhibitor HQNO, a structural analogue of the physiological electron donor. The redox potential of the catalytic lysine-coordinated high spin heme of NrfA is -50 ± 10 mV, z = 0.9. These values determined for the integral NrfH2A4 complex indicate that a driving force for a downhill electron transfer is ensured in this complex.

Complex I–complex II ratio strongly differs in various organs of Arabidopsis thaliana

In most studies, amounts of protein complexes of the oxidative phosphorylation (OXPHOS) system in different organs or tissues are quantified on the basis of isolated mitochondrial fractions. However, yield of mitochondrial isolations might differ with respect to tissue type due to varying efficiencies of cell disruption during organelle isolation procedures or due to tissue-specific properties of organelles. Here we report an immunological investigation on the ratio of the OXPHOS complexes in different tissues of Arabidopsis thaliana which is based on total protein fractions isolated from five Arabidopsis organs (leaves, stems, flowers, roots and seeds) and from callus. Antibodies were generated against one surface exposed subunit of each of the five OXPHOS complexes and used for systematic immunoblotting experiments. Amounts of all complexes are highest in flowers (likewise with respect to organ fresh weight or total protein content of the flower fraction). Relative amounts of protein complexes in all other fractions were determined with respect to their amounts in flowers. Our investigation reveals high relative amounts of complex I in green organs (leaves and stems) but much lower amounts in non-green organs (roots, callus tissue). In contrast, complex II only is represented by low relative amounts in green organs but by significantly higher amounts in non-green organs, especially in seeds. In fact, the complex I-complex II ratio differs by factor 37 between callus and leaf, indicating drastic differences in electron entry into the respiratory chain in these two fractions. Variation in amounts concerning complexes III, IV and V was less pronounced in different Arabidopsis tissues (quantification of complex V in leaves was not meaningful due to a cross-reaction of the antibody with the chloroplast form of this enzyme). Analyses were complemented by in gel activity measurements for the protein complexes of the OXPHOS system and comparative 2D blue native/SDS PAGE analyses using isolated mitochondria. We suggest that complex I has an especially important role in the context of photosynthesis which might be due to its indirect involvement in photorespiration and its numerous enzymatic side activities in plants.

Abstract 1137: Imp2 controls oxidative phosphorylation in glioblastoma cancer stem cells

Abstract Insulin-like growth factor 2 mRNA binding protein 2 (Imp2, IGF2BP2) is expressed during embryonic development in the brain and associated with several types of cancer. In preliminary studies we assessed Imp2 expression in normal brain and glial tumors. Immunohistochemistry revealed that Imp2 is present in 75% cases of the most malignant glial tumor, glioblastoma multiforme (GBM), whereas it is absent in low grade gliomas and in normal brain. GBM harbor a subpopulation of undifferentiated, slowly proliferating cells that correspond to the current definition of cancer stem cells (CSC). Analysis of these cells selected based on the CSC-associated CD133 marker expression, revealed that they express higher levels of Imp2 than their CD133-negative counterparts. Primary cultures of gliospheres that allow for CSC enrichment, also expressed high Imp2 levels. Imp2 depletion by shRNA significantly decreased GBM CSC ability to form spheres in single cell clonogenic assays and diminished their tumorigenic potential, suggesting that Imp2 is important for CSC maintenance. To investigate the role of Imp2 in CSC biology, Imp2 ribonucleoprotein complexes were purified and both Imp2-bound mRNA and proteins were analyzed, by microarray and mass spectrometry, respectively. Interestingly, both Imp2-bound RNA and Imp2-associated proteins were implicated in mitochondrial metabolism, in particular with oxidative phosphorylation (OxPhos). Involvement of Imp2 in mitochondrial metabolism was therefore tested. Depletion of Imp2 in GBM CSC impaired OxPhos, but did not affect other mitochondrial processes. Moreover, Imp2 knock-down decreased cellular ATP levels, suggesting that OxPhos is the main energy producing pathway in GBM CSC. Accordingly, inhibition of respiratory complex I with rotenone mimicked the Imp2 knock-down phenotype, as it diminished proliferation, sphere formation, oxygen consumption and ATP levels. In contrast, inhibition of anaerobic glycolysis, a pathway associated with cancer bioenergetics, did not affect GBM CSC. The mechanism by which Imp2 controls OxPhos may involve its protein interactors, among which we found three subunits of respiratory complex I, responsible for initiation of complex assembly. Imp2 depletion affects activity and assembly of respiratory complex I, suggesting that the interaction between Imp2 and these subunits is crucial for electron entry into the OxPhos chain. In addition, Imp2 may control cellular respiration by providing mRNA delivery. Purification of mitochondria-bound polyribosomes revealed that overexpression of Imp2 increases the amount of Imp2-bound mRNA in this fraction. These data imply that Imp2 delivers nuclear-encoded mRNA onto ribosomes attached to the mitochondrial surface, where co-translational insertion into mitochondrial membranes can occur. Our results have uncovered a novel function of the oncofetal protein Imp2 related to metabolic requirements of GBM CSC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1137. doi:1538-7445.AM2012-1137

Altered dopamine metabolism and increased vulnerability to MPTP in mice with partial deficiency of mitochondrial complex I in dopamine neurons

A variety of observations support the hypothesis that deficiency of complex I [reduced nicotinamide-adenine dinucleotide (NADH):ubiquinone oxidoreductase] of the mitochondrial respiratory chain plays a role in the pathophysiology of Parkinson's disease (PD). However, recent data from a study using mice with knockout of the complex I subunit NADH:ubiquinone oxidoreductase iron-sulfur protein 4 (Ndufs4) has challenged this concept as these mice show degeneration of non-dopamine neurons. In addition, primary dopamine (DA) neurons derived from such mice, reported to lack complex I activity, remain sensitive to toxins believed to act through inhibition of complex I. We tissue-specifically disrupted the Ndufs4 gene in mouse heart and found an apparent severe deficiency of complex I activity in disrupted mitochondria, whereas oxidation of substrates that result in entry of electrons at the level of complex I was only mildly reduced in intact isolated heart mitochondria. Further analyses of detergent-solubilized mitochondria showed the mutant complex I to be unstable but capable of forming supercomplexes with complex I enzyme activity. The loss of Ndufs4 thus causes only a mild complex I deficiency in vivo. We proceeded to disrupt Ndufs4 in midbrain DA neurons and found no overt neurodegeneration, no loss of striatal innervation and no symptoms of Parkinsonism in tissue-specific knockout animals. However, DA homeostasis was abnormal with impaired DA release and increased levels of DA metabolites. Furthermore, Ndufs4 DA neuron knockouts were more vulnerable to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Taken together, these findings lend in vivo support to the hypothesis that complex I deficiency can contribute to the pathophysiology of PD.

A comparative assessment of mitochondrial function in epimastigotes and bloodstream trypomastigotes of Trypanosoma cruzi

Trypanosoma cruzi is a hemoflagellate protozoan that causes Chagas' disease. The life cycle of T. cruzi is complex and involves different evolutive forms that have to encounter different environmental conditions provided by the host. Herein, we performed a functional assessment of mitochondrial metabolism in the following two distinct evolutive forms of T. cruzi: the insect stage epimastigote and the freshly isolated bloodstream trypomastigote. We observed that in comparison to epimastigotes, bloodstream trypomastigotes facilitate the entry of electrons into the electron transport chain by increasing complex II-III activity. Interestingly, cytochrome c oxidase (CCO) activity and the expression of CCO subunit IV were reduced in bloodstream forms, creating an "electron bottleneck" that favored an increase in electron leakage and H(2)O(2) formation. We propose that the oxidative preconditioning provided by this mechanism confers protection to bloodstream trypomastigotes against the host immune system. In this scenario, mitochondrial remodeling during the T. cruzi life cycle may represent a key metabolic adaptation for parasite survival in different hosts.

Nightside polar rain aurora boundary gap and its applications for magnetotail reconnection

[1] Polar rain electrons with energy around 1 keV occasionally fill the polar cap ionosphere. Their energy flux is high enough to produce detectable auroral emissions, called polar rain aurora (PRA). We report a PRA event on 2 August 2002 and the estimation of the magnetotail reconnection location and size from the PRA boundary. The distinguishing feature of this event identified from the TIMED/GUVI data is an emission depletion gap between PRA and auroral oval. The size of the gap is about 2° in latitude and extended from the dusk sector (17:00 MLT) to the premidnight sector (22:00 MLT). This gap coincides with the electron flux gap in the DMSP electron energy flux data. The electron flux data show the existence of energy dispersion at the gap. These GUVI and DMSP observations support the idea that the gap is very likely due to a magnetic reconnection in the magnetotail that blocks the solar wind electron entry into the polar ionosphere. Based on the electron energy dispersion, the nightside X line is estimated to be at X = −50 to −60 RE, which is consistent with results from other studies. The gap extends from the nightside to the duskside and presumably to the dawnside. This new information suggests that the extension of the tail X line over the entire plasma sheet in the dawn-dusk direction under a strongly southward IMF (Bz ∼ −10 nT) and the curved nature of the X line.

Orientational Control over Nitrite Reductase on Modified Gold Electrode and Its Effects on the Interfacial Electron Transfer

Recently, studies have been reported in which fluorescently labeled redox proteins have been studied with a combination of spectroscopy and electrochemistry. In order to understand the effect of the dye on the protein-electrode interaction, voltammetry and surface analysis have been performed on protein films of dye-labeled and unlabeled forms of a cysteine-surface variant (L93C) and the wild type (wt) of the copper containing nitrite reductase (NiR) from Alcaligenes faecalis S6. The protein has been adsorbed onto gold electrodes modified with self-assembled monolayers (SAMs) made up of 6-mercaptohexanol (6-OH) and mixtures of various octanethiols. Electrochemical and surface-analytical techniques were utilized to explore the influence of the SAM composition on wt and L93C NiR enzyme activity and the orientation of the enzyme molecules with respect to the electrode/SAM. The unlabeled L93C NiR enzyme is only electroactive on mixed SAMs composed of positive 8-aminooctanethiol (8-NH(2)) and 8-mercaptooctanol (8-OH). No enzymatic activity is observed on SAMs consisting of pure 6-OH, 8-OH, or pure 8-NH(2). Modification of L93C NiR with the ATTO 565 dye resulted in enzymatic activity on SAMs of 6-OH, but not on SAMs of 8-OH. Quartz crystal microbalance with dissipation measurements show that well-ordered and rigid protein films (single orientation of the protein) are formed when NiR is electroactive. By contrast, electrode-NiR combinations for which no electrochemical activity is observed still have NiR adsorbed on the surfaces, but a less-structured and water-rich film is formed. For the unlabeled L93C NiR, bilayer formation is observed, suggesting that the Cys93 residue is orientated away from the surface and able to form disulfide bridges to a second layer of L93C NiR. The results indicate that interfacial electron transfer is only possible if the negatively charged surface patch surrounding the electron-entry site of NiR is directed toward the electrode. This can be achieved either by introducing positive charges in the SAM or, when the SAM does not carry a charge, by labeling the enzyme with an ATTO 565 dye, which has some hydrophobic character, close to the electron entry site of the NiR.

Two internal type II NADH dehydrogenases of Toxoplasma gondii are both required for optimal tachyzoite growth

In many apicomplexan parasites the entry of electrons from NADH into the electron transport chain is governed by type II NADH dehydrogenases (NDH2s) instead of a canonical complex I. Toxoplasma gondii expresses two NDH2 isoforms, TgNDH2-I and TgNDH2-II with no indication for stage-specific regulation. We dissected the orientation of both isoforms by using a split GFP assay and a protease protection assay after selective membrane permeabilization. The two approaches revealed that both TgNDH2 isoforms are internal enzymes facing with their active sites to the mitochondrial matrix. Single knockout mutants displayed a decreased replication rate and a reduced mitochondrial membrane potential, which were both more severe in the Tgndh2-II-deleted than in the Tgndh2-I-deleted mutant. Complementation with a myc-tagged, ectopic copy of the deleted gene restored the growth rate and the mitochondrial membrane potential. However, an overexpression of the remaining intact isoform could not restore the phenotype, suggesting that the two TgNDH2 isoforms are non-redundant and possess functional differences. Together, our studies indicate that although TgNDH2-I and TgNDH2-II are individually non-essential, the expression of both internal isoforms is required to maintain the mitochondrial physiology in T. gondii tachyzoites.

Complex I of the Mitochondrial Electron Transport Chain is Dysfunctional in the Early Embryonic Heart

Proper cardiac function is crucial to ensure embryonic survival. The heart is the first organ to become functional during embryonic development, with the onset of beating at 8 days post fertilization (E8). Within two days of this (E10.5), blood begins to circulate providing nutrients to the developing embryo. Early defects in the embryonic heart can result in embryonic death or severe deformities leading to death during or shortly following birth. Although structural cardiac anomalies rarely cause demise, functional cardiac defects are much more devastating in utero.In the adult heart, mitochondria play an important role in proper function. Mitochondrial dysfunction can result in cardiac dysfunction and eventually death. While mitochondrial function is well studied in the adult heart, which relies on complex I as its primary source of electron entry, little is known about mitochondrial function in the developing embryonic heart.Data generated in this lab show that at the early stages of embryonic development (E9.5) mitochondrial membrane potential (Δψm) is low, and the potential for increased oxidative stress is high. A minimal change in Δψm is observed at E9.5 upon the addition of the complex I inhibitor rotenone. This observation is contrasted in E13.5 myocytes, which exhibit a higher sensitivity of Δψm to rotenone. In concert, these data suggest that at E9.5 complex I of the mitochondrial electron transport chain is non-functional. This study employs established bioenergetic and mitochondrial proteomic techniques, which have been adapted and applied in whole embryonic hearts and cardiomyocytes during cardiac organogenesis (E9.5, E11.5, E13.5) to support the hypothesis that at early stages of embryonic cardiac development, mitochondrial function is limited due to an immature complex I, thus resulting in decreased Δψm and increased potential for oxidative stress.

Proteins in biomimetic membranes: promises and facts

Different planar lipid bilayers are considered, which are designed as model systems for membrane proteins in a functionally active form. Tethered bilayer lipid membranes (tBLMs) were investigated in our group based on peptide- or oligo-oxy-ethylene (OEO) tethers. Peptide-tethered BLMs were shown to incorporate large proteins such as cytochrome c oxidase (CcO) isolated from bovine heart and F0F1 ATPase from chloroplasts, whereas OEO-tBLMs were designed to incorporate ion carriers such as valinomycin and small channel proteins such as melittin and gramicidin. The activity of these proteins could be demonstrated. However, a quantitative treatment was not feasible. Therefore, we have concentrated our efforts on a relatively new approach, the protein-tethered bilayer lipid membrane (ptBLM), which uses the protein itself as the essential building block. With the example of cytochrome c oxidase (CcO) from R. sphaeroides with His-tags attached to subunits I and II the protein was immobilized in opposite orientations and reconstituted into the respective ptBLMs. Direct electron transfer from an electrode into the enzyme could be measured by electrochemical and spectro-electrochemical methods when the enzyme was immobilized with the electron entry side directed toward the electrode. The uptake of 4 electrons per molecule CcO measured under anaerobic conditions was treated by rigorous electrochemical theory to follow a sequential, or ECCC mechanism. When the enzyme was exposed to an oxygenated solution an amplified current density indicated catalytic turnover. As far as spectro-electrochemistry was concerned, the most promising results were obtained by electrochemically controlled surface-enhanced infrared absorption spectroscopy (SEIRAS). Potentiometric titrations followed by SEIRAS indicated significant conformational changes of the protein as a function of potential. The enzyme could even be shown to adopt two different conformational states, a non-activated and an activated state before and after it was allowed to undergo catalytic turnover. Differences between the two states could be demonstrated particularly using 2D autocorrelation maps.

Molecular Basis for Directional Electron Transfer

Biological macromolecules involved in electron transfer reactions display chains of closely packed redox cofactors when long distances must be bridged. This is a consequence of the need to maintain a rate of transfer compatible with metabolic activity in the framework of the exponential decay of electron tunneling with distance. In this work intermolecular electron transfer was studied in kinetic experiments performed with the small tetraheme cytochrome from Shewanella oneidensis MR-1 and from Shewanella frigidimarina NCIMB400 using non-physiological redox partners. This choice allowed the effect of specific recognition and docking to be eliminated from the measured rates. The results were analyzed with a kinetic model that uses the extensive thermodynamic characterization of these proteins reported in the literature to discriminate the kinetic contribution of each heme to the overall rate of electron transfer. This analysis shows that, in this redox chain that spans 23 A, the kinetic properties of the individual hemes establish a functional specificity for each redox center. This functional specificity combined with the thermodynamic properties of these soluble proteins ensures directional electron flow within the cytochrome even outside of the context of a functioning respiratory chain.

Direct electrochemistry of dinuclear CuA fragment from cytochrome c oxidase of Thermus thermophilus at surfactant modified glassy carbon electrode

Abstract Cytochrome c oxidase is ubiquitous enzyme involved in the terminal step of respiratory electron transfer process. The unique binuclear copper center containing bis-dithiolato bridges form a valance delocalized [Cu 1.5+ –Cu 1.5+ ] state of the metal center located at the subunit II of cytochrome c oxidase. This metal center acts as the electron entry site of the enzyme and accepts electrons from cytochrome c. Direct electrochemistry of this binuclear copper center containing the water soluble protein obtained by genetically truncating the membrane bound part of the subunit II from Thermus thermophilus was achieved by favorable orientation of the protein on glassy carbon electrode surface promoting efficient electron transfer in the presence of various surfactants. Very reproducible, Nernstian responses are obtained with Cu A . The redox potential and the electrochemical response were enhanced prominently in case of cationic surfactant CTAB indicating that the nature of the surfactant has a significant effect on the microenvironment of the protein–electrode interface. The results have been used to understand the mechanism of electron transfer from cytochrome c to the copper center during the enzymatic reaction.

Intramolecular Electron Transfer in Pseudomonas aeruginosa cd1 Nitrite Reductase: Thermodynamics and Kinetics

The cd 1 nitrite reductases, which catalyze the reduction of nitrite to nitric oxide, are homodimers of 60 kDa subunits, each containing one heme- c and one heme- d 1. Heme- c is the electron entry site, whereas heme- d 1 constitutes the catalytic center. The 3D structure of Pseudomonas aeruginosa nitrite reductase has been determined in both fully oxidized and reduced states. Intramolecular electron transfer (ET), between c and d 1 hemes is an essential step in the catalytic cycle. In earlier studies of the Pseudomonas stutzeri enzyme, we observed that a marked negative cooperativity is controlling this internal ET step. In this study we have investigated the internal ET in the wild-type and His369Ala mutant of P. aeruginosa nitrite reductases and have observed similar cooperativity to that of the Pseudomonas stutzeri enzyme. Heme- c was initially reduced, in an essentially diffusion-controlled bimolecular process, followed by unimolecular electron equilibration between the c and d 1 hemes ( k ET = 4.3 s −1 and K = 1.4 at 298K, pH 7.0). In the case of the mutant, the latter ET rate was faster by almost one order of magnitude. Moreover, the internal ET rate dropped (by ∼30-fold) as the level of reduction increased in both the WT and the His mutant. Equilibrium standard enthalpy and entropy changes and activation parameters of this ET process were determined. We concluded that negative cooperativity is a common feature among the cd 1 nitrite reductases, and we discuss this control based on the available 3D structure of the wild-type and the H369A mutant, in the reduced and oxidized states.

Cyanobacterial cytochrome cM: Probing its role as electron donor for Cu A of cytochrome c oxidase

It is well known that efficient functioning of photosynthetic (PET) and respiratory electron transport (RET) in cyanobacteria requires the presence of either cytochrome c 6 (Cyt c 6) or plastocyanin (PC). By contrast, the interaction of an additional redox carrier, cytochrome c M (Cyt c M), with either PET or RET is still under discussion. Here, we focus on the (putative) role of Cyt c M in cyanobacterial respiration. It is demonstrated that genes encoding the main terminal oxidase (cytochrome c oxidase, COX) and cytochrome c M are found in all 44 totally or partially sequenced cyanobacteria (except one strain). In order to check whether Cyt c M can act as electron donor to COX, we investigated the intermolecular electron transfer kinetics between Cyt c M and the soluble Cu A domain (i.e. the donor binding and electron entry site) of subunit II of COX. Both proteins from Synechocystis PCC6803 were expressed heterologously in E. coli. The forward and the reverse electron transfer reactions were studied yielding apparent bimolecular rate constants of (2.4 ± 0.1) × 10 5 M − 1 s − 1 and (9.6 ± 0.4) × 10 3 M − 1 s − 1 (5 mM phosphate buffer, pH 7, 50 mM KCl). A comparative analysis with Cyt c 6 and PC demonstrates that Cyt c M functions as electron donor to Cu A as efficiently as Cyt c 6 but more efficient than PC. Furthermore, we demonstrate the association of Cyt c M with the cytoplasmic and thylakoid membrane fractions by immunobloting and discuss the potential role of Cyt c M as electron donor for COX under stress conditions.

Polar rain gradients and field‐aligned polar cap potentials

ACE SWEPAM measurements of solar wind field‐aligned electrons have been compared with simultaneous measurements of polar rain electrons precipitating over the polar cap and detected by DMSP spacecraft. Such comparisons allow investigation of cross‐polar‐cap gradients in the intensity of otherwise‐steady polar rain. The generally good agreement of the distribution functions, f, from the two data sources confirms that direct entry of solar electrons along open field lines is indeed the cause of polar rain. The agreement between the data sets is typically best on the side of the polar cap with most intense polar rain but the DMSP f's in less intense regions can be brought into agreement with ACE measurements by shifting all energies by a fixed amounts that range from tens to several hundred eV. In most cases these shifts are positive which implies that field‐aligned potentials of these amounts exist on polar cap field lines which tend to retard the entry of electrons and produce the observed gradients. These retarding potentials undoubtedly appear in order to prevent the entry of low‐energy electrons and maintain charge quasi‐neutrality that would otherwise be violated since most tailward flowing magnetosheath ions are unable to follow polar rain electrons down to the polar cap. In more limited regions near the boundary of the polar cap there is sometimes evidence for field‐aligned potentials of the opposite sign that accelerate polar rain electrons. A solar electron burst is also studied and it is concluded that electrons from such bursts can enter the magnetotail and precipitate in the same manner as polar rain.

Inhibition of bacterial oxidases by formamide and analogs

The enzymatic activity of Paracoccus denitrificans cytochrome c oxidase (COX) and Escherichia coli cytochrome b(o) ubiquinol oxidase (QOX) was determined in the presence of formamide, N,N-dimethyl formamide and N,N-dimethyl acetamide. Formamide was found to inhibit the enzyme activity of the oxidases most significantly, whereas the other two compounds inhibited the activity to a lesser extent. The effects of formamide and analogs on enzyme activity were very similar for COX and QOX, indicating that the mechanism of inhibition might be the same for both of these oxidases. The inhibition kinetics followed a non-competitive mechanism. Studies using proteoliposomes of COX and QOX containing the electron entry site of the enzyme directed towards the outside of the vesicles showed that the effect of inhibition by formamide was higher when the inhibitor was present on the outside of the proteoliposome compared to when it was present only in the aqueous core. This indicates that inhibition of enzyme activity by formamide possibly predominantly involves blocking of the water exit pathway in the oxidases.

Monte Carlo Modeling of the Wall Effect in Cylindrical Tissue-Equivalent Proportional Counters from Heavy Ions

Heavy ions constitute a significant part of radiation in space, they are used for radiation therapy and laboratory experiments with accelerators. The useful instrument for measurement of radiation dose and investigation radiation quality factor for heavy ions is tissue equivalent proportional counter (TEPC). It was found previously [1,2] that response of spherical wall TEPC shows distortions at the wall/cavity interface due to enhanced entry of secondary electrons into the sensitive volume of counter. In this study it was theoretically investigated whether similar effect is expected in cylinder TEPC. The Monte Carlo code [1] originally designed for spherical TEPC was updated by extension with cylindrical shape of internal gas cavity. It was tested the following three geometries of irradiation: ion tracks are parallel-, perpendicular to cylindrical detector axis and µ-randomness superposition. The coordinates of track segments in the wall and gas cavity were calculated using stochastic track structure code PITS to score the energy deposition in the counter. The internal gas cavity with a 0.635 cm radius, 1.27 cm height contains tissue equivalent gas at 7.87 e-5 g/cm**3 density and wall plastic density - 1.0 g/cm**3. Liquid water cross sections were chosen as a target to approximate the plastic and gas. The stochastic tracks of 20-Ne ions with energy 46 MeV/nucleon and LET=167 keV/µm were used for modeling of TEPC response. The linear energy for three mentioned beam geometries was calculated and plotted against impact parameter which is the perpendicular distance from the center of counter to the ion track. In contract to spherical counter [1,2] for the cylindrical TEPC was found no clear occurrence of the peak at the interface between the wall and the gas for all three tested geometries. Nothing but in the case when ion beam is parallel to TEPC axis the weak effect as a small height peak succeeded resulted from grazing events. This findings lead to the conclusion that wall effect like [1] observed in spherical TEPC hardly is expected in experiments with cylindrical proportional counters. References. 1. Rademacher S. E. et al. (1998) Radiat. Res. 149, 387-395. 2. Nikjoo H., Khvostunov I. K., Cucinotta F. A. (2002) Radiat. Res. 157, 435-445.