In cellular respiration, electron transfer from the breakdown of foodstuff is coupled to the formation of an electrochemical proton gradient. This is accomplished through proton translocation by respiratory complexes, and the proton gradient is subsequently used e.g. to drive ATP production. Consequently, proton- and electron-transfer reactions through the hydrophobic interior of membrane proteins are central to cellular respiration. In this thesis, proton- and electron transfer through an aa3-type terminal oxidase, cytochrome c oxidase (CytcO) from Rhodobacter sphaeroides, have been studied with the aim of understanding the molecular proton-transfer machinery of this proton pump. In the catalytic site of CytcO the electrons combine with protons and the terminal electron acceptor O2 to form water in an exergonic reaction that drives proton pumping. Therefore, CytcO must transfer both protons that are pumped and protons for the oxygen chemistry through its interior. This is done through its two proton-transfer pathways, termed the D pathway and the K pathway. Our studies have shown that the protons pumped during oxidation of CytcO are taken through the D pathway, and that this process does not require a functional K pathway. Furthermore, our data suggests that the K pathway is used for charge compensation of electron transfer to the catalytic site, but only in the A2 → P3 state transition. Our data also show that the water molecules identified in the crystal structures of CytcO play an important role in proton transfer through the D pathway. Finally, the effects of liposome reconstitution of CytcO on D-pathway proton transfer were investigated. The results suggest that the membrane modulates the rates of proton transfer through the D pathway, and also influences the extent of electron transfer between redox-active sites CuA and heme a.

Cytochromes c are electron transfer proteins in which haem is covalently attached to the polypeptide chain via thioether bonds formed from thiol groups of the two cysteines and the two vinyl groups of haem. This attachment is a post translational process and in many species of bacteria as many as approximately twelve gene products, the functions of which are largely unknown, are required. In Gram-negative bacteria the assembly of the c-type cytochromes occurs in the periplasm. Cytochrome c₅₅₂ from the thermophilic organism Hydrogenobacter thermophilus is known to be expressed in the cytoplasm of Escherichia coli. This unique example of cytoplasmic assembly of a c-type cytochrome has previously been postulated to result from insertion of haem into the folded apoform of the cytochrome followed by non-catalysed attachment of the haem. This postulate is supported by the present work which has shown that the cytoplasmic assembly of this cytochrome c₅₅₂ continues in the absence of the E. coli ccm genes which are needed for 'normal' c-type cytochrome assembly in that organism. Attempts to test the postulate of spontaneous assembly of the cytochrome c₅₅₂ with in vitro experiments require large amounts of cytochrome c₅₅₂ and its apo protein. A number of procedures for preparing these proteins were investigated. Although a T7-based expression produced lower amounts than was expected, its use led to detection of the apo form of cytochrome c₅₅₂ in E. coli. It was shown that this apoform has some secondary structure, whereas mitochondrial apocytochrome c has a random coil conformation. This observation is consistent with, but does not prove, the postulate for cytochrome c₅₅₂ assembly. It was unexpectedly found that a strain of E. coli that produces abnormally large amounts of its endogenous c-type cytochromes also made large amounts of cytoplasmic cytochrome c₅₅₂. NMR studies on this material are consistent with a single and 'normal' attachment of the haem to the polypeptide. Thus the unusual cytoplasmic assembly was not different from the usual periplasmic assembly that occurs in the H. thermophilus itself. In E. coli there is a periplasmic cytochrome b₅₆₂ that is presumed to acquire its haem in the periplasm. Some of the ccm genes, required for c-type cytochrome assembly, are postulated to code for a system that transports haem to the periplasm. Cytochrome b- ₅₆₂ synthesis was not blocked by the absence of these genes. This implies that haem provision for cytochrome b₅₆₂ synthesis occurs independently of the ccm system. Apocytochrome b<sub>562 could be detected in E. coli with the ratio apo:holo being higher in a strain that produces c-type cytochromes to relatively low levels. It is suggested that the synthesis of both cytochrome b₅₆₂ and c-type cytochromes is at least partly a reflection of the rate of production of haem by the cells.

Cytochrome c oxidase (CcO), the terminal enzyme in the electron transport chain, couples proton pumping to the reduction of dioxygen into water. The coupling mechanism remains to be elucidated. Previous studies have identified several mutations within CcO's primary proton uptake pathway (the D-channel) that decouple proton pumping from redox activity. Here, I examine the molecular basis for decoupling in single and double mutants of highly conserved residues, D132 and N139, in order to gain insight into the coupling mechanism. In particular, I use molecular dynamics and free energy simulations of a new, unconstrained model of bacterial CcO embedded in a solvated lipid bilayer to investigate how such mutants affect functional hydration and ionic selectivity in the D-channel. Results support earlier mechanistic insights obtained in our laboratory from simplified molecular models and predict a new, testable hypothesis by which cations such as K+ may inhibit proton pumping in charged mutants of N139.

Cytochrome c oxidase (CcO), the terminal enzyme in the electron transport chain, couples proton pumping to the reduction of dioxygen into water. The coupling mechanism remains to be elucidated. Previous studies have identified several mutations within CcO's primary proton uptake pathway (the D-channel) that decouple proton pumping from redox activity. Here, I examine the molecular basis for decoupling in single and double mutants of highly conserved residues, D132 and N139, in order to gain insight into the coupling mechanism. In particular, I use molecular dynamics and free energy simulations of a new, unconstrained model of bacterial CcO embedded in a solvated lipid bilayer to investigate how such mutants affect functional hydration and ionic selectivity in the D-channel. Results support earlier mechanistic insights obtained in our laboratory from simplified molecular models and predict a new, testable hypothesis by which cations such as K+ may inhibit proton pumping in charged mutants of N139.

Thesis (M.S.)--Brigham Young University. Dept. of Biology, 2007. / Includes bibliographical references.

A interação do citocromo c com a cardiolipina ocorre por interações eletrostáticas e hidrofóbicas. A formação do complexo citocromo c/ cardiolipina promove uma pequena mudança estrutural na proteína, que proporciona atividade peroxidásica ao citocromo c e consequentemente capacidade de oxidar substratos orgânicos, incluindo a cardiolipina. A oxidação da cardiolipina acompanhada da inserção de um grupo peróxido vem sendo relacionada à perda da interação hidrofóbica entre o complexo citocromo c/cardiolipina, que resulta no desligamento do citocromo c da membrana e na sua saída do espaço intermembranas para o citosol, onde essa proteína induz a cascata de apoptose. Neste trabalho foi avaliada a ligação do citocromo c a lipossomos contendo cardiolipina oxidada e a reatividade desta proteína com o mono-hidroperóxido da cardiolipina (TLCL(OOH)1) presente na membrana. Nossos dados mostraram que ocorre uma diminuição significativa na ligação do citocromo c a membrana oxidadas apenas quando 100% da cardiolipina presente na membrana está na forma de TLCL(OOH)1, condição que extrapolaria o que seria esperado para o sistema biológico. Análises por SDS-PAGE revelaram que o citocromo c sofre agregação na presença de membranas contendo TLCL(OOH)1, indicando que a proteína reage com este peróxido. De fato, determinamos a velocidade de reação do citocromo c com o TLCL(OOH)1 e com hidroperóxido do ácido linoléico, inseridos em membrana contendo cardiolipina (9,58 ± 0,16 x 102 M-1.s-1 e 6,91 ± 0,30 x 102 M-1.s-1, respectivamente). As velocidades de reação com os peróxidos de lipídio foram pelo menos 10 vezes superiores à velocidade medida com o peróxido de hidrogênio (5,91 ± 0,18 x101 M-1.s-1). Assim, mostramos que o citocromo c liga-se à membrana contendo hidroperóxido de cardiolipina e que reage com o mesmo promovendo a formação de agregado protéico de alto peso molecular / The interaction of cytochrome c with cardiolipin is promoted by electrostatic and hydrophobic interactions. The cytochrome c / cardiolipin complex formation causes structural changes in the protein that activates cytochrome c peroxidase activity, giving it the ability to oxidize organic substrates, including cardiolipin. The oxidation of cardiolipin coupled with a peroxide group insertion has been related to the loss of hydrophobic interactions between the cytochrome c / cardiolipin complex, resulting in cytochrome c release from the membrane and in its translocation from intermembranes space to cytosol, where this protein induces apoptosis cascade. In this work the binding of cytochrome c to liposomes containing oxidized cardiolipin and its reactivity with the membrane mono-hydroperoxides (TLCL(OOH)1) were evaluated. Our data showed a significant decrease in cytochrome c binding to oxidized membranes only when 100% of the membrane cardiolipin is in the TLCL(OOH)1 form, a condition that would extrapolate the expected concentrations that would be found in a biological system. SDS-PAGE analysis revealed that cytochrome c undergoes aggregation in the presence of membranes containing TLCL(OOH)1, indicating that this protein reacts with the peroxide. In fact, we determined the rate of cytochrome c reaction with TLCL(OOH)1 and linoleic acid hydroperoxide inserted into cardiolipin containing membranes (9.58 ± 0.16 x 102 M-1s-1 and 6.91 ± 0.30 x 102 M-1s-1,respectively). The reaction rates obtained with lipid peroxides were at least 10 times higher than that obtained with hydrogen peroxide (5.91 ± 0.18 x 101 M-1s-1).Thus we show that cytochrome c binds to membrane containing cardiolipin hydroperoxides and reacts with it promoting the formation of high molecular weight protein aggregates.

A associação lipídio/citocromo c é importante e deve ser estudada, pois repercute na atividade peroxidática da proteína abordada e pode contribuir para o processo apoptótico, ou morte programada da célula, e também desempenha um papel significativo na cadeia respiratória. A natureza e a especificidade da interação do citocromo c com bicamadas lipídicas têm sido bastante investigadas ultimamente, mas informações detalhadas e precisas sobre tais assuntos ainda não existem. É aceito que ocorre primeiramente uma interação eletrostática entre a proteína citocromo c e as membranas fosfolipídicas. Em seguida, há uma interação hidrofóbica. Entretanto, ainda não é bem compreendido o papel da cadeia fosfolipídica. A associação do citocromo c com membranas lipídicas induz mudanças no estado de spin do átomo de ferro. A interação entre as vesículas carregadas e o citocromo c induz mudanças estruturais na proteína, as quais são refletidas no seu centro ativo, ou grupo heme. As mudanças do campo cristalino no sítio do ferro hemínico de forte para fraco são acompanhadas por mudanças do estado de spin de baixo para alto, respectivamente. Neste trabalho, estuda-se sistematicamente a natureza da interação entre o citocromo c e a cadeia fosfolipídica. As mudanças estruturais no grupo heme foram correlacionadas com a natureza do lipídio, ou seja, com a carga da cabeça e com o tamanho e o tipo da cadeia fosfolipídica. Foram utilizados treze lipídios diferentes, naturais e sintetizados, com cabeças polares negativas e neutras e com cadeias carbônicas saturadas e insaturadas de diferentes comprimentos. Para tal investigação, utilizamos as técnicas: Ressonância Paramagnética Eletrônica (RPE) Onda Contínua (CW) e Pulsada (PW) e Dicroísmo Circular Magnético (MCD). As técnicas enunciadas avaliam as mudanças de estado de spin e a simetria do citocromo c nos seus estados férrico e ferroso. A interação lipoprotéica lipídio/citocromo c foi avaliada com lipídios diferentes, inclusive com o lipossomo PCPECL, que mimetiza a membrana interna da mitocôndria nos eucariontes. A partir dos resultados experimentais, sugerimos um modelo para esse tipo de associação. / This association lipid/cytochrome c is interesting to study in order to understand the peroxidase activity of this protein, that plays an important role in the respiratory chain and in the apoptosis process or the programmed cell death. The nature and specificity of the interaction of cytochrome c with lipid bilayers have been major goals in recent studies, but detailed information on that issue is not yet widely available. In this regard, it is generally accepted that the electrostatic interaction is an important factor in the association of cytochrome c with phospholipid membranes, followed by a hydrophobic interaction. However, the role played by the phospholipid chain is not well understood. The association of cytochrome c with negative membranes induces a change in the heme iron spin state. The interaction between the charged vesicles and cytochrome c leads to structural changes in the active central or heme group. The changing of the crystalline field of the heme iron from strong to weak is accompanied by spin states changes from low to high spin, respectively. These facts concerned us to investigate more systematically the nature of the interaction between cytochrome c and the phospholipid chains. The lipid-induced effects in the heme iron crystalline field are correlated to the nature of the charged head group and to the size and type of the phospholipid chain. Thirteen different lipids, nature and synthetic, were used, with negative and neutra1 polar head group and saturated and unsaturated acyl chains with different length. This work investigates the change of heme iron spin state and symmetry of ferric cytochrome c using Continuous Wave (CW) and pulsed (PW) Electron Paramagnetic Resonance (EPR) and Magnetic Circular Dichroism (MCD) techniques. These techniques analyze the spin state change and the symmetry of the iron cytochrome c in its ferric and ferrous states. The effect of the different lipids were analyzed, including PCPECL membrane that mimetics the inner mitocondrial membrane in eukaryotes.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Os complexos trans-[Ru(CNpy)(NH3)4(1,4-dt)]2+ (CNpy = 4-Cianopiridina, 1,4- dt = 1,4-ditiano), trans-[Ru(CNpy)(NH3)4(pyS)]2+ (pyS = 4-mercaptopiridina) e trans- [Ru(CNpy)(NH3)4(Tio)]2+ (Tio = tionicotinamida) foram sintetizados e caracterizados por Cromatografia LÃquida de Alta EficiÃncia, anÃlise elementar, eletroquÃmica e espectroscopias eletrÃnica e vibracional. Os espectros de absorÃÃo eletrÃnica e os resultados de eletroquÃmica evidenciaram uma interaÃÃo do tipo &#960; back-bonding do centro metÃlico RuII para os ligantes piridÃnicos. Os valores dos potenciais formais de meia onda (E1/2) observados para os complexos trans-[Ru(CNpy)(NH3)4(1,4-dt)]2+, trans-[Ru(CNpy)(NH3)4(pyS)]2+ e trans-[Ru(CNpy)(NH3)4(Tio)]2+: 1,00, 0,67 e 1,03 V vs ENH, respectivamente, indicam a estabilizaÃÃo do estado reduzido do centro metÃlico em relaÃÃo ao complexo de partida trans-[Ru(CNpy)(NH3)4(OH2)]2+ (E1/2 = 0,35 V vs ENH). Esse resultado indica, tambÃm, a forte capacidade &#960; retiradora do ligante CNpy que, no caso do complexo trans-[Ru(CNpy)(NH3)4(1,4-dt)]2+, à tÃo intensa que facilita comparativamente o processo de oxidaÃÃo do centro metÃlico. Podese concluir, portanto, que a capacidade &#960; retiradora dos ligantes CNpy, pyS e Tio à similar. O deslocamento na freqÃÃncia de estiramento da ligaÃÃo C&#8801;N do ligante Cnpy livre de 2239 para &#8764;2200 cm-1 apÃs coordenaÃÃo ao centro metÃlico [Ru(NH3)4]2+ nos espectros vibracionais indica que a coordenaÃÃo do ligante CNpy ocorre pelo grupamento nitrila. Para os ligantes pyS e Tio, a observaÃÃo das bandas atribuÃdas aos &#957;C=S e &#957;SH, respectivamente, indica que a coordenaÃÃo ocorre pelo Ãtomo de nitrogÃnio do anel piridÃnico. Os espectros SERS ex situ das superfÃcies modificadas com os compostos isolados permitem a classificaÃÃo destes como molÃculas modificadoras bifuncionais, onde o Ãtomo de enxofre dos ligantes pyS, 1,4-dt e Tio atuam como sÃtio de adsorÃÃo e o Ãtomo de nitrogÃnio piridÃnico do ligante CNpy atua como grupo funcional terminal. AlÃm disso, os resultados SERS sugerem uma conformaÃÃo trans para o complexo trans-[Ru(CNpy)(NH3)4(pyS)]2+ e gauche para os complexos trans-[Ru(CNpy)(NH3)4(1,4-dt)]2+ e trans-[Ru(CNpy)(NH3)4(Tio)]2+ sobre ouro. A dependÃncia nas intensidades das bandas com o potencial aplicado aos eletrodos modificados ilustra o efeito &#960; back-bonding dos Ãons metÃlicos dos adsorbatos inorgÃnicos, uma vez que observa-se uma relaÃÃo direta com os valores dos potenciais formais de meia onda dos complexos estudados. Os valores dos potenciais de dessorÃÃo redutiva observados para as SAMs dos complexos isolados refletem a capacidade &#960; retiradora do ligante CNpy que se encontra na posiÃÃo para em relaÃÃo ao sÃtio de adsorÃÃo. Esse efeito à bem mais intenso no complexo trans- [Ru(CNpy)(NH3)4(1,4-dt)]2+, uma vez que a capacidade de interaÃÃo &#960; back-bonding do ligante 1,4-dt à significativamente menor que as dos ligantes pyS e Tio. Sugere-se, para esse complexo, uma oxidaÃÃo parcial do centro metÃlico o que implica em uma intensificaÃÃo da interaÃÃo &#963; com o ligante 1,4-dt (RuII-S) e, conseqÃentemente, um enfraquecimento da ligaÃÃo Au-S. A reaÃÃo hTE da metaloproteÃna cyt c foi observada em valor consistente com seu estado in natura indicando que as SAMs estudadas acessam satisfatoriamente o processo hTE do cyt c alÃm de evitar a desnaturaÃÃo desta proteÃna. A forma das curvas voltamÃtricas, entretanto, apresentou uma dependÃncia com a configuraÃÃo dos complexos sobre a superfÃcie. Curvas de melhor definiÃÃo foram obtidas com a SAM do complexo trans-[Ru(CNpy)(NH3)4(pyS)]2+ que apresenta uma configuraÃÃo trans em relaÃÃo à superfÃcie. / Trans-[Ru(CNpy)(NH3)4(L)]2+ type complexes, where CNpy (4- Cyanopyridine), L= 1,4-dt (1,4-dithiane), pyS (4-mercaptopyridine), and Tio (thionicotinamide), were synthesized and characterized by HPLC, microanalysis, eletrochemistry and vibrational and eletronic spectroscopies. The eletrochemical and eletronic absorption results are indicative of a &#960; back-bonding interaction, L(n&#960;*) &#8592; RuII(d&#960;) for which L is the pyS, CNpy and Tio ligands. The half-wave formal potentials (E1/2) observed for the trans-[Ru(CNpy)(NH3)4(pyS)]2+, trans- [Ru(CNpy)(NH3)4(1,4-dt)]2+ and trans-[Ru(CNpy)(NH3)4(Tio)]2+ complexes: 1.00, 0.67 and 1.03 V vs NHE, respectively, are indicative of the stabilization of the metal in the reduced state comparatively to the start complex, trans-[Ru(CNpy)(NH3)4(OH2)]2+ (E1/2 = 0.35 V vs NHE). This result indicates, also, the strong &#960; withdraw capability of the CNpy ligand. The shift observed for the &#957;C&#8801;N mode from 2239 cm-1 in the vibrational spectra of the CNpy free ligand to &#8764;2200 cm-1 upon coordination suggests that the coordination occurs through the nitrile fragment. For the pyS and Tio ligands, the observation of the signals assigned to the &#957;C=S and &#957;SH modes, respectively, in the vibrational spectra os the complexes indicates that the coordination occurs through the nitrogen atom os the pyridine ring. The SERS ex situ spectra of the gold surfaces modified with the isolated complexes allow the classification of these species as binfunctional molecules in which the sulfur atom of the pyS, 1,4-dt and Tio ligands is the adsorption site and the nitrogen atom of the CNpy pyridine ring is the functional terminal group. Also, the SERS results point for a trans conformation for the trans- [Ru(CNpy)(NH3)4(pyS)]2+ complex and for a gauche configuration for the trans- [Ru(CNpy)(NH3)4(1,4-dt)]2+ and trans-[Ru(CNpy)(NH3)4(Tio)]2+ complexes on gold. The intensity dependence of some signals in the SERS spectra with the applie potential reflects the &#960; back-bonding effect on surface since this behavior is observed to be strongly related to the E1/2 values of the complexes. The reductive desorption potentials of the SAMs formed with the isolated compounds reflects the &#960; withdraw capability of the CNpy ligand. This effect is much more intense for the trans-[Ru(CNpy)(NH3)4(1,4-dt)]2+ complex since the &#960; back-bonding interaction of the 1,4-dt ligand is lower than the CNpy, pyS and Tio ligands. The heterogeneous electron transfer reaction of the cytochrome c metalloprotein was satisfactorily assessed with the SAMs formed by the isolated complexes (E1/2 = 0.05 V vs Ag&#9168;AgCl&#9168;Cl-). The Best voltammetric response was observed with the SAM of the trans- [Ru(CNpy)(NH3)4(pyS)]2+ complex due to the trans conformation on surface.

In neurons, mitochondrial quantity and basal cellular respiration are maintained with age, but alterations in other key functions and quantities make these cells susceptible to the pathology of age-related neurodegenerative disease. We observed age-related decreases in cytochrome C, cardiolipin, cytochrome C oxidase (CCO) function, and glutamate response that render cells less capable of responding to stress. Rescue experiments showed that estrogen is a promising treatment in restoring neuron function with age. After finding key differences in CCO, we examined the electron transport chain more closely and found age-related deficits in quantity or function for each individual complex. Our experiments support a lack of endogenous substrates or a failure of upstream complexes to transport electrons to complex IV with age, ultimately leading to age-related neurodegeneration. Reactive oxygen species production may add to the problem by degrading macromolecules such as nucleic acid, cardiolipin, and proteins. Increased ROS may also lead to a redox imbalance in the neuron, reducing the potential for energy production. Also, epigenetic controls such as DNA methylation, histone acetylation ubiquitination and phosphorylation that persist in culture independent of aging hormone levels, vasculature, and immune system may be partly responsible for the observed age-related deficiencies as has been previously observed in aging human muscle (Ronn et al., 2008). This compelling cumulative evidence suggests an age-related deficiency in electron transport via quinones from complexes I to III, and age-related deficiencies in substrates, cofactors, and quantity or function for complex IV. These studies add to the growing body of evidence that dysfunction in the enzyme complexes of the electron transport chain lead to neurodegeneration in senescence-related diseases. In an attempt to integrate our age-related findings with Alzheimer's Disease (AD) pathology, we sequentially isolated the electron transport chain complexes using selective mitochondrial inhibitors in cortical neurons removed from the 3xTg-AD mouse model, which harbors mutations in the PS1, APPSwe and tauP301L genes and follows the proposed temporal development of human AD pathology (Oddo et al., 2003a; 2003b). Overall, we did not detect 3xTg-AD cortical neuron deficits at the four electron transport complexes of mitochondria or in NAD(P)H oxidase (NOX), an extramitochondrial oxygen consumer and regulator of NAD(P)+/NAD(P)H homeostasis (Morre et al., 2000).

Heme containing proteins and their reactivity play a central role in biological systems; they have a vast range of functions including electron transfer, catalysis, and respiration. Cytochromes c and heme c containing proteins have been used widely as model systems to understand how structure and dynamics lead toward function. In this thesis, a variety of biophysical methods are used to investigate two heme c containing model systems to gain insight into how redox potential and reactivity are modulated through changes in the local environment. Mitochondrial cytochrome c undergoes several pH dependent conformational rearrangements that involve different heme ligation and have associated changes in redox potential. Under basic conditions (pH greater than 8), the axial methionine (Met) residue is replaced by one of several nitrogen based ligands, usually a nearby lysine residue, and is coined the "alkaline transition". It is accompanied by a large downward shift in redox potential. The functional utility of this conformational change is not fully understood however it is strongly implicated in the signaling cascade for apoptosis. Bacterial monoheme cytochromes c exhibit similar phenomenological Met-loss behavior as a function of electrode material. In Chapter 2 we utilize Hydrogenobacter thermophilus cytochrome c552 as a model system for the assessment of redox thermodynamics and changes in redox potential associated with the Met-loss form. In Chapter 3 we extend our investigation to homologous cytochromes c. Bacterial cytochrome c peroxidases catalyze the two-electron reduction of hydrogen peroxide to water utilizing cytochrome c as an endogenous electron donor. Chapter 4 describes the first recombinant construct of the diheme Nitrosomonas europaea cytochrome c peroxidase (Ne CCP); a defining family member of constitutively active cytochrome c peroxidases. A variety of biophysical techniques were used to confirm similarity between the recombinant Ne CCP and native enzyme. Chapter 5 extends our investigation to the role of constitutively conserved glutamine and glutamic acid residues within the active site, and two conserved tryptophan residues; the first situated between hemes and the second distal to the active site. In Chapter 6, stopped flow spectroscopy is used to investigate the first intermediates of the Ne CCP catalytic mechanism.