Oxygen reduction and proton translocation by cytochrome c oxidase

Energy conversion by living organisms is central dogma of bioenergetics. The effectiveness of the energy extraction by aerobic organisms is much greater than by anaerobic ones. In aerobic organisms the final stage of energy conversion occurs in respiratory chain that is located in the inner membrane of mitochondria or cell membrane of some aerobic bacteria. The terminal complex of the respiratory chain is cytochrome c oxidase (CcO) - the subject of this study. The primary function of CcO is to reduce oxygen to water. For this, CcO accepts electrons from a small soluble enzyme cytochrome c from one side of the membrane and protons from another side. Moreover, CcO translocates protons across the membrane. Both oxygen reduction and proton translocation contributes to generation of transmembrane electrochemical gradient that is used for ATP synthesis and different types of work in the cell. Although the structure of CcO is defined with a relatively high atomic resolution (1.8 Å), its function can hardly be elucidated from the structure. The electron transfer route within CcO and its steps are very well defined. Meanwhile, the proton transfer roots were predicted from the site-specific mutagenesis and later proved by X-ray crystallography, however, the more strong proof of the players of the proton translocation machine is still required. In this work we developed new methods to study CcO function based on FTIR (Fourier Transform Infrared) spectroscopy. Mainly with use of these methods we answered several questions that were controversial for many years: [i] the donor of H+ for dioxygen bond splitting was identified and [ii] the protolytic transitions of Glu-278 one of the key amino acid in proton translocation mechanism was shown for the first time.

A quantum chemical investigation of the metal centres in cytochrome c oxidase

Quantum effects are often of key importance for the function of biological systems at molecular level. Cellular respiration, where energy is extracted from the reduction of molecular oxygen to water, is no exception. In this work, the end station of the electron transport chain in mitochondria, cytochrome c oxidase, is investigated using quantum chemical methodology. Cytochrome c oxidase contains two haems, haem a and haem a3. Haem a3, with its copper companion, CuB, is involved in the final reduction of oxygen into water. This binuclear centre receives the necessary electrons from haem a. Haem a, in turn, receives its electrons from a copper ion pair in the vicinity, called CuA. Density functional theory (DFT) has been used to clarify the charge and spin distributions of haem a, as well as changes in these during redox activity. Upon reduction, the added electron is shown to be evenly distributed over the entire haem structure, important for the accommodation of the prosthetic group within the protein. At the same time, the spin distribution of the open-shell oxidised state is more localised to the central iron. The exact spin density distribution has been disputed in the literature, however, different experiments indicating different distributions of the unpaired electron. The apparent contradiction is shown to be due to the false assumption of a unit amount of unpaired electron density; in fact, the oxidised state has about 1.3 unpaired electrons. The validity of the DFT results have been corroborated by wave function based coupled cluster calculations. Point charges, for use in classical force field based simulations, have been parameterised for the four metal centres, using a newly developed methodology. In the procedure, the subsystem for which point charges are to be obtained, is surrounded by an outer region, with the purpose of stabilising the inner region, both electronically and structurally. Finally, the possibility of vibrational promotion of the electron transfer step between haem a and a3 has been investigated. Calculating the full vibrational spectra, at DFT level, of a combined model of the two haems, revealed several normal modes that do shift electron density between the haems. The magnitude of the shift was found to be moderate, at most. The proposed mechanism could have an assisting role in the electron transfer, which still seems to be dominated by electron tunnelling.

Redox-linked proton transfer by cytochrome c oxidase

The respiratory chain is found in the inner mitochondrial membrane of higher organisms and in the plasma membrane of many bacteria. It consists of several membrane-spanning enzymes, which conserve the energy that is liberated from the degradation of food molecules as an electrochemical proton gradient across the membrane. The proton gradient can later be utilized by the cell for different energy requiring processes, e.g. ATP production, cellular motion or active transport of ions. The difference in proton concentration between the two sides of the membrane is a result of the translocation of protons by the enzymes of the respiratory chain, from the negatively charged (N-side) to the positively charged side (P-side) of the lipid bilayer, against the proton concentration gradient. The endergonic proton transfer is driven by the flow of electrons through the enzymes of the respiratory chain, from low redox-potential electron donors to acceptors of higher potential, and ultimately to oxygen. Cytochrome c oxidase is the last enzyme in the respiratory chain and catalyzes the reduction of dioxygen to water. The redox reaction is coupled to proton transport across the membrane by a yet unresolved mechanism. Cytochrome c oxidase has two proton-conducting pathways through which protons are taken up to the interior part of the enzyme from the N-side of the membrane. The K-pathway transfers merely substrate protons, which are consumed in the process of water formation at the catalytic site. The D-pathway transfers both substrate protons and protons that are pumped to the P-side of the membrane. This thesis focuses on the role of two conserved amino acids in proton translocation by cytochrome c oxidase, glutamate 278 and tryptophan 164. Glu278 is located at the end of the D-pathway and is thought to constitute the branching point for substrate and pumped protons. In this work, it was shown that although Glu278 has an important role in the proton transfer mechanism, its presence is not an obligatory requirement. Alternative structural solutions in the area around Glu278, much like the ones present in some distantly related heme-copper oxidases, could in the absence of Glu278 support the formation of a long hydrogen-bonded water chain through which proton transfer from the D-pathway to the catalytic site is possible. The other studied amino acid, Trp164, is hydrogen bonded to the ∆-propionate of heme a3 of the catalytic site. Mutation of this amino acid showed that it may be involved in regulation of proton access to a proton acceptor, a pump site, from which the proton later is expelled to the P-side of the membrane. The ion pair that is formed by the ∆-propionate of heme a3 and arginine 473 is likely to form a gate-like structure, which regulates proton mobility to the P-side of the membrane. The same gate may also be part of an exit path through which water molecules produced at the catalytically active site are removed towards the external side of the membrane. Time-resolved optical and electrometrical experiments with the Trp164 to phenylalanine mutant revealed a so far undetected step in the proton pumping mechanism. During the A to PR transition of the catalytic cycle, a proton is transferred from Glu278 to the pump site, located somewhere in the vicinity of the ∆-propionate of heme a3. A mechanism for proton pumping by cytochrome c oxidase is proposed on the basis of the presented results and the mechanism is discussed in relation to some relevant experimental data. A common proton pumping mechanism for all members of the heme-copper oxidase family is moreover considered.

Theoretical Studies on Coupled Electron and Proton Transfer in Cytochrome c Oxidase

The complexity of life is based on an effective energy transduction machinery, which has evolved during the last 3.5 billion years. In aerobic life, the utilization of the high oxidizing potential of molecular oxygen powers this machinery. Oxygen is safely reduced by a membrane bound enzyme, cytochrome c oxidase (CcO), to produce an electrochemical proton gradient over the mitochondrial or bacterial membrane. This gradient is used for energy-requiring reactions such as synthesis of ATP by F0F1-ATPase and active transport. In this thesis, the molecular mechanism by which CcO couples the oxygen reduction chemistry to proton-pumping has been studied by theoretical computer simulations. By building both classical and quantum mechanical model systems based on the X-ray structure of CcO from Bos taurus, the dynamics and energetics of the system were studied in different intermediate states of the enzyme. As a result of this work, a mechanism was suggested by which CcO can prevent protons from leaking backwards in proton-pumping. The use and activation of two proton conducting channels were also enlightened together with a mechanism by which CcO sorts the chemical protons from pumped protons. The latter problem is referred to as the gating mechanism of CcO, and has remained a challenge in the bioenergetics field for more than three decades. Furthermore, a new method for deriving charge parameters for classical simulations of complex metalloenzymes was developed.

Cytochrome P450-mediated drug interactions affecting lidocaine

Lidocaine is a widely used local anaesthetic agent that also has anti-arrhythmic effects. It is classified as a type Ib anti-arrhythmic agent and is used to treat ventricular tachycardia or ventricular fibrillation. Lidocaine is eliminated mainly by metabolism, and less than 5% is excreted unchanged in urine. Lidocaine is a drug with a medium to high extraction ratio, and its bioavailability is about 30%. Based on in vitro studies, the earlier understanding was that CYP3A4 is the major cytochrome P450 (CYP) enzyme involved in the metabolism of lidocaine. When this work was initiated, there was little human data on the effect of inhibitors of CYP enzymes on the pharmacokinetics of lidocaine. Because lidocaine has a low therapeutic index, medications that significantly inhibit lidocaine clearance (CL) could increase the risk of toxicity. These studies investigated the effects of some clinically important CYP1A2 and CYP3A4 inhibitors on the pharmacokinetics of lidocaine administered by different routes. All of the studies were randomized, double-blind, placebo-controlled cross-over studies in two or three phases in healthy volunteers. Pretreatment with clinically relevant doses of CYP3A4 inhibitors erythromycin and itraconazole or CYP1A2 inhibitors fluvoxamine and ciprofloxacin was followed by a single dose of lidocaine. Blood samples were collected to determine the pharmacokinetic parameters of lidocaine and its main metabolites monoethylglycinexylidide (MEGX) and 3-hydroxylidocaine (3-OH-lidocaine). Itraconazole and erythromycin had virtually no effect on the pharmacokinetics of intravenous lidocaine, but erythromycin slightly prolonged the elimination half-life (t½) of lidocaine (Study I). When lidocaine was taken orally, both erythromycin and itraconazole increased the peak concentration (Cmax) and the area under the concentration-time curve (AUC) of lidocaine by 40-70% (Study II). Compared with placebo and itraconazole, erythromycin increased the Cmax and the AUC of MEGX by 40-70% when lidocaine was given intravenously or orally (Studies I and II). The pharmacokinetics of inhaled lidocaine was unaffected by concomitant administration of itraconazole (Study III). Fluvoxamine reduced the CL of intravenous lidocaine by 41% and prolonged the t½ of lidocaine by 35%. The mean AUC of lidocaine increased 1.7-fold (Study IV). After oral administration of lidocaine, the mean AUC of lidocaine in-creased 3-fold and the Cmax 2.2-fold by fluvoxamine (Study V). During the pretreatment with fluvoxamine combined with erythromycin, the CL of intravenous lidocaine was 53% smaller than during placebo and 21% smaller than during fluvoxamine alone. The t½ of lidocaine was significantly longer during the combination phase than during the placebo or fluvoxamine phase. The mean AUC of intravenous lidocaine increased 2.3-fold and the Cmax 1.4-fold (Study IV). After oral administration of lidocaine, the mean AUC of lidocaine increased 3.6-fold and the Cmax 2.5-fold by concomitant fluvoxamine and erythromycin. The t½ of oral lidocaine was significantly longer during the combination phase than during the placebo (Study V). When lidocaine was given intravenously, the combination of fluvoxamine and erythromycin prolonged the t½ of MEGX by 59% (Study IV). Compared with placebo, ciprofloxacin increased the mean Cmax and AUC of intravenous lidocaine by 12% and 26%, respectively. The mean plasma CL of lidocaine was reduced by 22% and its t½ prolonged by 7% (Study VI). These studies clarify the principal role of CYP1A2 and suggest only a modest role of CYP3A4 in the elimination of lidocaine in vivo. The inhibition of CYP1A2 by fluvoxamine considerably reduces the elimination of lidocaine. Concomitant use of fluvoxamine and the CYP3A4 inhibitor erythromycin further increases lidocaine concentrations. The clinical implication of this work is that clinicians should be aware of the potentially increased toxicity of lidocaine when used together with inhibitors of CYP1A2 and particularly with the combination of drugs inhibiting both CYP1A2 and CYP3A4 enzymes.

Drag på parnassen. Två sextiotalsstudier. : Del I: Medelklass med mänskligt ansikte. Medelklassradikalism i fyra romaner av Christer Kihlman, Jarl Sjöblom, Marianne Alopaeus och Ulla-Lena Lundberg. Del II: Modernistdebatten. Sak, person och fält i en finlandssvensk litteraturdebatt år 1965.

The objective of my dissertation Pull (or Draught, or Moves) at the Parnassus , is to provide a deeper understanding of Nordic Middle Class radicalism of the 1960 s as featured in Finland-Swedish literature. My approach is cultural materialist in a broad sense; social class is regarded a crucial aspect of the contents and contexts of the novels and literary discussions explored. In the first volume, Middle Class With A Human Face , novels by Christer Kihlman, Jarl Sjöblom, Marianne Alopaeus, and Ulla-Lena Lundberg, respectively, are read from the points of view of place, emotion, and power. The term "cryptotope" is used to designate the hidden places found to play an important role in all of these four narratives. Also, the "chronotope of the provincial small town", described by Mikhail Bakhtin in 1938, is exemplified in Kihlman s satirical novel, as is the chronotope of of war (Algeria, Vietnam) in those of Alopaeus and Lundberg s. All the four novels signal changes in the way general "scripts of emotions", e.g. jealousy, are handled and described. The power relations in the novels are also read, with reference to Michel Foucault. As the protagonists in two of them work as journalists, a critical discussion about media and Bourgeois hegemony is found; the term "repressive legitimation" is created to grasp these patterns of manipulation. The Modernist Debate , part II of the study, concerns a literary discussion between mainly Finland-Swedish authors and critics. Essayist Johannes Salminen (40) provided much of the fuel for the debate in 1963, questioning the relevance to contemporary life of the Finland-Swedish modernist tradition of the 1910 s and 1920 s. In 1965, a group of younger authors and critics, including poet Claes Andersson (28), followed up this critique in a debate taking place mainly in the newspaper Vasabladet. Poets Rabbe Enckell (62), Bo Carpelan (39) and others defended a timeless poetry. This debate is contextualized and the changing literary field is analyzed using concepts provided by sociologist Pierre Bourdieu. In the thesis, the historical moment of Middle Class radicalism with a human face is regarded a temporary luxury that new social groups could afford themselves, as long as they were knocking over the statues and symbols of the Old Bourgeoisie. This is not to say that all components of the Sixties strategy have lost their power. Some of them have survived and even grown, others remain latent in the gene bank of utopias, waiting for new moments of change.

Expression of cytochrome P450 enzymes and metabolism of timolol in human ocular tissue

Glaucoma is a group of progressive optic neuropathies causing irreversible blindness if not diagnosed and treated in the early state of progression. Disease is often, but not always, associated with increased intraocular pressure (IOP), which is also the most important risk factor for glaucoma. Ophthlamic timolol preparations have been used for decades to lower increased intraocular pressure (IOP). Timolol is locally well tolerated but may cause e.g. cardiovascular and pulmonary adverse effects due to systemic absorption. It has been reported that approximately 80% of a topically administered eye drop is systemically absorbed. However, only limited information is available on timolol metabolism in the liver or especially in the human eye. The aim of this work was to investigate metabolism of timolol in human liver and human ocular tissues. The expression of drug metabolizing cytochrome P450 (CYP) enzymes in the human ciliary epithelial cells was studied. The metabolism of timolol and the interaction potential of timolol with other commercially available medicines were investigated in vitro using different liver preparations. The absorption of timolol to the aqueous humor from two commercially available products: 0.1% eye gel and 0.5% eye drops and the presence of timolol metabolites in the aqueous humor were investigated in a clinical trial. Timolol was confirmed to be metabolized mainly by CYP2D6 as previously suggested. Potent CYP2D6 inhibitors especially fluoxetine, paroxetine and quinidine inhibited the metabolism of timolol. The inhibition may be of clinical significance in patients using ophthalmic timolol products. CYP1A1 and CYP1B1 mRNAs were expressed in the human ciliary epithelial cells. CYP1B1 was also expressed at protein level and the expression was strongly induced by a known potent CYP1B1 inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The CYP1B1 induction is suggested to be mediated by aryl hydrocarbon receptor (AHR). Low levels of CYP2D6 mRNA splice variants were expressed in the human ciliary epithelial cells and very low levels of timolol metabolites were detected in the human aqueous humor. It seems that negligible amount of CYP2D6 protein is expressed in the human ocular tissues. Timolol 0.1% eye gel leads to aqueous humor concentration high enough to achieve therapeutic effect. Inter-individual variation in concentrations is low and intraocular as well as systemic safety can be increased when using this product with lower timolol concentration instead of timolol 0.5% eye drops.

Cervical headgear in Class II division 1 correction in children : Academic Dissertation

Class II division 1 malocclusion occurs in 3.5 to 13 percent of 7 12 year-old children. It is the most common reason for orthodontic treatment in Finland. Correction is most commonly performed using headgear treatment. The aim of this study was to investigate the effects of cervical headgear treatment on dentition, facial skeletal and soft tissue growth, and upper airway structure, in children. 65 schoolchildren, 36 boys and 29 girls were studied. At the onset of treatment a mean age was 9.3 (range 6.6 12.4) years. All the children were consequently referred to an orthodontist because of Class II division 1 malocclusion. The included children had protrusive maxilla and an overjet of more than 2mm (3 to 11 mm). The children were treated with a Kloehn-type cervical headgear as the only appliance until Class I first molar relationships were achieved. The essential features of the headgear were cervical strong pulling forces, a long upward bent outer bow, and an expanded inner bow. Dental casts and lateral and posteroanterior cephalograms were taken before and after the treatment. The results were compared to a historical, cross-sectional Finnish cohort or to historical, age- and sex-matched normal Class I controls. The Class I first molar relationships were achieved in all the treated children. The mean treatment time was 1.7 (range 0.3-3.1) years. Phase 2 treatments were needed in 52% of the children, most often because of excess overjet or overbite. The treatment decreased maxillary protrusion by inhibiting alveolar forward growth, while the rest of the maxilla and mandible followed normal growth. The palate rotated anteriorly downward. The expansion of the inner bow of the headgear induced widening of the maxilla, nasal cavity, and the upper and lower dental arches. Class II malocclusion was associated with narrower oro- and hypopharyngeal space than in the Class I normal controls. The treatment increased the retropalatal airway space, while the rest of the airway remained unaffected. The facial profile improved esthetically, while the facial convexity decreased. Facial soft tissues masked the facial skeletal convexity, and the soft tissue changes were smaller than skeletal changes. In conclusion, the headgear treatment with the expanded inner bow may be used as an easy and simple method for Class II correction in growing children.

Studying the diurnal and seasonal acclimation of photosystem Ii using chlorophyll-a fluorescence

A small fraction of the energy absorbed in the light reactions of photosynthesis is re-emitted as chlorophyll-a fluorescence. Chlorophyll-a fluorescence and photochemistry compete for excitation energy in photosystem II (PSII). Therefore, changes in the photochemical capacity can be detected through analysis of chlorophyll fluorescence. Chlorophyll fluorescence techniques have been widely used to follow the diurnal (fast), and the seasonal (slow) acclimation in the energy partitioning between photochemical and non-photochemical processes in PSII. Energy partitioning in PSII estimated through chlorophyll fluorescence can be used as a proxy of the plant physiological status, and measured at different spatial and temporal scales. However, a number of technical and theoretical limitations still limit the use of chlorophyll fluorescence data for the study of the acclimation of PSII. The aim of this Thesis was to study the diurnal and seasonal acclimation of PSII in field conditions through the development and testing of new chlorophyll fluorescence-based tools, overcoming these limitations. A new model capable of following the fast acclimation of PSII to rapid fluctuations in light intensity was developed. The model was used to study the rapid acclimation in the electron transport rate under fluctuating light. Additionally, new chlorophyll fluorescence parameters were developed for estimating the seasonal acclimation in the sustained rate constant of thermal energy dissipation and photochemistry. The parameters were used to quantitatively evaluate the effect of light and temperature on the seasonal acclimation of PSII. The results indicated that light environment not only affected the degree but also the kinetics of response of the acclimation to temperature, which was attributed to differences in the structural organization of PSII during seasonal acclimation. Furthermore, zeaxanthin-facilitated thermal dissipation appeared to be the main mechanisms modulating the fraction of absorbed energy being dissipated thermally during winter in field Scots pine. Finally, the integration between diurnal and seasonal acclimation mechanisms was studied using a recently developed instrument MONI-PAM (Walz GmbH, Germany) capable of continuously monitoring the energy partitioning in PSII.

Asymmetrical flow field-flow fractionation in the study of water-soluble macromolecules

Asymmetrical flow field-flow fractionation (AsFlFFF) was constructed, and its applicability to industrial, biochemical, and pharmaceutical applications was studied. The effect of several parameters, such as pH, ionic strength, temperature and the reactants mixing ratios on the particle sizes, molar masses, and the formation of aggregates of macromolecules was determined by AsFlFFF. In the case of industrial application AsFlFFF proved to be a valuable tool in the characterization of the hydrodynamic particle sizes, molar masses and phase transition behavior of various poly(N-isopropylacrylamide) (PNIPAM) polymers as a function of viscosity and phase transition temperatures. The effect of sodium chloride salt and the molar ratio of cationic and anionic polyelectrolytes on the hydrodynamic particle sizes of poly (methacryloxyethyl trimethylammonium chloride) and poly (ethylene oxide)-block-poly (sodium methacrylate) and their complexes were studied. The particle sizes of PNIPAM polymers, and polyelectrolyte complexes measured by AsFlFFF were in agreement with those obtained by dynamic light scattering. The molar masses of PNIPAM polymers obtained by AsFlFFF and size exclusion chromatography agreed also well. In addition, AsFlFFF proved to be a practical technique in thermo responsive behavior studies of polymers at temperatures up to about 50 oC. The suitability of AsFlFFF for biological, biomedical, and pharmaceutical applications was proved, upon studying the lipid-protein/peptide interactions, and the stability of liposomes at different temperatures. AsFlFFF was applied to the studies on the hydrophobic and electrostatic interactions between cytochrome c (a basic peripheral protein) and anionic lipid, and oleic acid, and sodium dodecyl sulphate surfactant. A miniaturized AsFlFFF constructed in this study was exploited in the elucidation of the effect of copper (II), pH, ionic strength, and vortexing on the particle sizes of low-density lipoproteins.

Christianity and Christian Identity in the Gospel of Philip (NHC II,3)

Previous scholarship has often maintained that the Gospel of Philip is a collection of Valentinian teachings. In the present study, however, the text is read as a whole and placed into a broader context by searching for parallels from other early Christian texts. Although the Valentinian Christian identity of the Gospel of Philip is not questioned, it is read alongside those texts traditionally labelled as "mainstream Christian". It is obvious from the account of Irenaeus that the boundaries between the Valentinians and other Christians were not as clear or fixed as he probably would have hoped. This study analyzes the Valentinian Christian Gospel of Philip from two points of view: how the text constructs the Christian identity and what kind of Christianity it exemplifies. Firstly, it is observed how the author of the Gospel of Philip places himself and his Christian readers among the early Christianities of the period by emphasizing the common history and Christian features but building especially on particular texts and traditions. Secondly, it is noted how the Christian nature of an individual develops according to the Gospel of Philip. The identity of an individual is built and strengthened through rituals, experiences and teaching. Thirdly, the categorizations, attributes, beliefs and behaviour associated on the one hand with the "insiders", the true Christians, and, on the other, with outsiders in the Gospel of Philip, are analyzed using social identity theory the insiders and outsiders are described through stereotyping in the text. Overall, the study implies that the Gospel of Philip strongly emphasizes spiritual progress and transformation. Rather than depicting the Valentinians as the perfect Christians, it underlines their need for constant change and improvement. Although the author seeks to clearly distinguish the insiders from the outsiders, the boundaries of the categories are in fact fluid in the Gospel of Philip. Outsiders can become insiders and the insiders are also in danger of falling out again.