Molecular basis of intramolecular electron transfer in sulfite-oxidizing enzymes is revealed by high resolution structure of a heterodimeric complex of the catalytic molybdopterin subunit and a c-type cytochrome subunit

Sulfite-oxidizing molybdoenzymes convert the highly reactive and therefore toxic sulfite to sulfate and have been identified in insects, animals, plants, and bacteria. Although the well studied enzymes from higher animals serve to detoxify sulfite that arises from the catabolism of sulfur-containing amino acids, the bacterial enzymes have a central role in converting sulfite formed during dissimilatory oxidation of reduced sulfur compounds. Here we describe the structure of the Starkeya novella sulfite dehydrogenase, a heterodimeric complex of the catalytic molybdopterin subunit and a c-type cytochrome subunit, that reveals the molecular mechanism of intramolecular electron transfer in sulfite-oxidizing enzymes. The close approach of the two redox centers in the protein complex (Mo-Fe distance 16.6 angstrom) allows for rapid electron transfer via tunnelling or aided by the protein environment. The high resolution structure of the complex has allowed the identification of potential through-bond pathways for electron transfer including a direct link via Arg-55A and/or an aromatic-mediated pathway. A potential site of electron transfer to an external acceptor cytochrome c was also identified on the SorB subunit on the opposite side to the interaction with the catalytic SorA subunit.

Intra- vs intermolecular photoinduced electron transfer reactions of a macrocyclic donor-acceptor dyad

The synthesis, structural characterization, and photophysical behavior of a 14-membered tetraazamacrocycle with pendant 4-dimethylaminobenzyl (DMAB) and 9-anthracenylmethyl groups is reported (L-3, 6-((9-anthracenylmethyl)amino)-trans-6,13-dimethyl-13-((4-dimethylaminobenzyl)amino)-1,4,8,11-tetraaza-cyclotetradecane). In its free base form, this compound displays rapid intramolecular photoinduced electron transfer (PET) quenching of the anthracene emission, with both the secondary amines and the DMAB group capable of acting as electron donors. When complexed with Zn(II), the characteristic fluorescence of the anthracene chromophore is restored as the former of these pathways is deactivated by coordination. Importantly, it is shown that the DMAB group, which remains uncoordinated and PET active, acts only very weakly to quench emission, by comparison to the behavior of a model Zn complex lacking the pendant DMAB group, [ZnL2](2+) (Chart 1). By contrast, Stern-Volmer analysis of intermolecular quenching of [ZnL2](2+) by N,N-dimethylaniline (DMA) has shown that this reaction is diffusion limited. Hence, the pivotal role of the bridge in influencing intramolecular PET is highlighted.

The influence of defects on the electron-transfer and magnetic properties of RbxMn[Fe(CN)6]y·zH2O

The synthesis and detailed characterization of a few samples of the compound RbMn[Fe(CN)]·zHO are described. The composition of the materials significantly depends on the applied preparative conditions. Analysis of spectroscopic results (FTIR, Raman, Fe Mössbauer, XPS) and X-ray powder-diffraction data yielded a further assessment of the difference in structural features in terms of the amount of Fe(CN)6 vacancies and the associated number of water molecules. The characteristic individual magnetic behavior, as well as the metal-to-metal charge-transfer capabilities of the various samples, could be related to significant changes within the structures that appear to be associated with the synthetic method used.

Applications of Photoinduced Electron Transfer Chemistry: Photoremovable Protecting Groups and Carbon Dioxide Conversion

Traditional organic chemistry has long been dominated by ground state thermal reactions. The alternative to this is excited state chemistry, which uses light to drive chemical transformations. There is considerable interest in using this clean renewable energy source due to concerns surrounding the combustion byproducts associated with the consumption of fossil fuels. The work presented in this text will focus on the use of light (both ultraviolet and visible) for the following quantitative chemical transformations: (1) the release of compounds containing carboxylic acid and alcohol functional groups and (2) the conversion of carbon dioxide into other useable chemicals. Chapters 1-3 will introduce and explore the use of photoremovable protecting groups (PPGs) for the spatiotemporal control of molecular concentrations. Two new PPGs are discussed, the 2,2,2-tribromoethoxy group for the protection of carboxylic acids and the 9-phenyl-9-tritylone group for the protection of alcohols. Fundamental interest in the factors that affect C–X bond breaking has driven the work presented in this text for the release of carboxylic acid substrates. Product analysis from the UV photolysis of 2,2,2-tribromoethyl-(2′-phenylacetate) in various solvents results in the formation of H–atom abstraction products as well as the release of phenylacetic acid. The deprotection of alcohols is realized through the use of UV or visible light photolysis of 9-phenyl-9-tritylone ethers. Central to this study is the use of photoinduced electron transfer chemistry for the generation of ion diradicals capable of undergoing bond-breaking chemistry leading to the release of the alcohol substrates. Chapters 4 and 5 will explore the use of N-heterocyclic carbenes (NHCs) as a catalyst for the photochemical reduction of carbon dioxide. Previous experiments have demonstrated that NHCs can add to CO2 to form stable zwitterionic species known as N-heterocylic-2-carboxylates (NHC–CO2). Work presented in this text illustrate that the stability of these species is highly dependent on solvent polarity, consistent with a lengthening of the imidazolium to carbon dioxide bond (CNHC–CCO2). Furthermore, these adducts interact with excited state electron donors resulting in the generation of ion diradicals capable of converting carbon dioxide into formic acid.

Structural and electron self-exchange rate variations in isomeric (hexaamine)cobalt(III/II) complexes

The syntheses and characterisation of the new macrocyclic hexaamine trans-(5(S),7(S),12(R),14(R)-tetramethyl)-1,4,8,11-tetraazacyclotetradecane-6,13-diamine (L-6) and its Co-III complex are reported. The X-ray crystal structural analyses of [CoL6]Cl-2(ClO4) [monoclinic, space group C2/c, a = 16.468(3) Angstrom, b = 9.7156(7) Angstrom, c = 15.070(3) Angstrom, beta = 119.431(8)degrees, Z = 4] and the closely related cis-diamino-substituted macrocyclic complex [CoL2](ClO4)(3) . 2H(2)O (L-2 = cis-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane-6,13-diamine) [orthorhombic, space group Pna2(1), a = 16.8220(8) Angstrom, b = 10.416(2) Angstrom, c = 14.219(3) Angstrom, Z = 4] reveal significant variations in the observed Co-N bond lengths and coordination geometries, which may be attributed to the trans or cis disposition of the pendent primary amines. The Co-III/II self-exchange electron transfer rate constants for these and other closely related hexaamines have been determined, and variations of some 2 orders of magnitude are found between pairs of trans and cis isomeric Co-III complexes.

Differentiated chemical reactivity of nanoparticles toward DTT

RATIONALE: Induction of oxidative stress and impairment of the antioxidant defense are considered important biological responses following nanoparticle (NP) exposure. The acellular in vitro dithiothreitol (DTT) assay is proposed to measure the oxidative potential of NP. In addition, DTT can be considered as a model compound of sulfur containing antioxidants. The objective of this work is to evaluate the surface reactivity in solution of a NP panel toward DTT. METHOD: The NP panel was composed of four carbonaceous particles, six types of metal oxides and silver with primary size ranged from 7 to 300 nm. Suspensions were prepared in surfactant solution with 30 min sonication. DTT was used as reductant to evaluate the oxidative properties of the different NP. The determination of the NP ability to catalyze electron transfer from DTT to oxygen was carried out as described in Sauvain et al., Nanotoxicology, 2008, 2:3, 121−129. RESULTS: All the carbonaceous NP catalyzed the oxidation of DTT by oxygen following the mass based order: carbon black > diesel exhaust particle > nanotubes > fullerene. A contrasting reactivity was observed for the metallic NP. Except for nickel oxide and metallic silver, which reacted similarly to the carbonaceous NP, all other metal oxides hindered the oxidation of DTT by oxygen, with ZnO being the most effective one. CONCLUSIONS : DTT was stabilized against oxidation in the presence of metal oxide NP in the solution. This suggests that different chemical interactions take place compared with carbonaceous NP. To explain these differences, we hypothesize that DTT could form complexes with the metal oxide surface (or dissolved metal ions), rendering it less susceptible to oxidation. By analogy, such a process could be thought to apply in biological systems with sulfur−containing antioxidants, reducing their buffer capacity. Such NP could thus contribute to oxidative stress by an alternative mechanism.

Triggering the generation of an iron(IV)-oxo compound and its reactivity toward sulfides by RuII photocatalysis

The preparation of [FeIV(O)(MePy2tacn)]2+ (2, MePy2tacn = N-methyl-N,N-bis(2-picolyl)-1,4,7-triazacyclononane) by reaction of [FeII(MePy2tacn)(solvent)]2+ (1) and PhIO in CH3CN and its full characterization are described. This compound can also be prepared photochemically from its iron(II) precursor by irradiation at 447 nm in the presence of catalytic amounts of [Ru II(bpy)3]2+ as photosensitizer and a sacrificial electron acceptor (Na2S2O8). Remarkably, the rate of the reaction of the photochemically prepared compound 2 toward sulfides increases 150-fold under irradiation, and 2 is partially regenerated after the sulfide has been consumed; hence, the process can be repeated several times. The origin of this rate enhancement has been established by studying the reaction of chemically generated compound 2 with sulfides under different conditions, which demonstrated that both light and [Ru II(bpy)3]2+ are necessary for the observed increase in the reaction rate. A combination of nanosecond time-resolved absorption spectroscopy with laser pulse excitation and other mechanistic studies has led to the conclusion that an electron transfer mechanism is the most plausible explanation for the observed rate enhancement. According to this mechanism, the in-situ-generated [RuIII(bpy)3] 3+ oxidizes the sulfide to form the corresponding radical cation, which is eventually oxidized by 2 to the corresponding sulfoxide

Reactivity characteristics of cytochrome c, cytochrome oxidase and the electrostatic cytochrome c - cytochrome oxidase complex /

The mechanism whereby cytochrome £ oxidase catalyses elec-. tron transfer from cytochrome £ to oxygen remains an unsolved problem. Polarographic and spectrophotometric activity measurements of purified, particulate and soluble forms of beef heart mitochondrial cytochrome c oxidase presented in this thesis confirm the following characteristics of the steady-state kinetics with respect to cytochrome £: (1) oxidation of ferrocytochrome c is first order under all conditions. -(2) The relationship between sustrate concentration and velocity is of the Michaelis-Menten type over a limited range of substrate. concentrations at high ionic strength. (3) ~he reaction rate is independent from oxygen concentration until very low levels of oxygen. (4) "Biphasic" kinetic plots of enzyme activity as a function of substrate concentration are found when the range of cytochrome c concentrations is extended; the biphasicity ~ is more apparent in low ionic strength buffer. These results imply two binding sites for cytochrome £ on the oxidase; one of high affinity and one of low affinity with Km values of 1.0 pM and 3.0 pM, respectively, under low ionic strength conditions. (5) Inhibition of the enzymic rate by azide is non-c~mpetitive with respect to cytochrome £ under all conditions indicating an internal electron transfer step, and not binding or dissociation of £ from the enzyme is rate limiting. The "tight" binding of cytochrome '£ to cytochrome c oxidase is confirmed in column chromatographic experiments. The complex has a cytochrome £:oxidase ratio of 1.0 and is dissociated in media of high ionic strength. Stopped-flow spectrophotometric studies of the reduction of equimolar mixtures and complexes of cytochrome c and the oxidase were initiated in an attempt to assess the functional relevance of such a complex. Two alternative routes -for reduction of the oxidase, under conditions where the predominant species is the £ - aa3 complex, are postulated; (i) electron transfer via tightly bound cytochrome £, (ii) electron transfer via a small population of free cytochrome c interacting at the "loose" binding site implied from kinetic studies. It is impossible to conclude, based on the results obtained, which path is responsible for the reduction of cytochrome a. The rate of reduction by various reductants of free cytochrome £ in high and low ionic strength and of cytochrome £ electrostatically bound to cytochrome oxidase was investigated. Ascorbate, a negatively charged reagent, reduces free cytochrome £ with a rate constant dependent on ionic strength, whereas neutral reagents TMPD and DAD were relatively unaffected by ionic strength in their reduction of cytochrome c. The zwitterion cysteine behaved similarly to uncharged reductants DAD and TI~PD in exhibiting only a marginal response to ionic strength. Ascorbate reduces bound cytochrome £ only slowly, but DAD and TMPD reduce bound cytochrome £ rapidly. Reduction of cytochrome £ by DAD and TMPD in the £ - aa3 complex was enhanced lO-fold over DAD reduction of free £ and 4-fold over TMPD reduction of free c. Thus, the importance of ionic strength on the reactivity of cytochrome £ was observed with the general conclusion being that on the cytochrome £ molecule areas for anion (ie. phosphate) binding, ascorbate reduction and complexation to the oxidase overlap. The increased reducibility for bound cytochrome £ by reductants DAD and TMPD supports a suggested conformational change of electrostatically bound c compare.d to free .£. In addition, analysis of electron distribution between cytochromes £ and a in the complex suggest that the midpotential of cytochrome ~ changes with the redox state of the oxidase. Such evidence supports models of the oxidase which suggest interactions within the enzyme (or c - enzyme complex) result in altered midpoint potentials of the redox centers.

Synthesis, structure and reactivity of novel CuI, CuII and CuIII complexes containing triaza and hexaaza macrocyclic ligands

El treball de tesi s'emmarca dins del camp de la bioinorgànica, disciplina que estudia les propietats estructurals i de reactivitat dels centres actius dels enzims, servint-se de models síntètics de baix pes molecular per tal d'intentar reproduïr la reactivitat presentada per l'enzim i conèixer els mecanismes de reacció a nivell molecular que tenen lloc en els processos biològics.1 Més concretament el treball posa especial èmfasi en els processos d'activació d'oxigen molecular que tenen lloc en les metaloproteïnes de Coure del Tipus 3, com són l'hemocianina i la tirosinasa, ambdues presentant un complex dinuclear de Cu(I)) en el centre actiu de la forma reduïda, capaç d'activar l'O2 cap a espècies de tipus peròxid.2 Un altre camp d'interès ha estat l'estudi dels processos d'activació d'enllaços C-H no activats en hidrocarburs, tant per la seva importàcia a nivell industrial com per comprendre els mecanismes intrínsecs d'aquesta activació a través de metalls de trancisió.3,4 Durant el treball de tesi presentat s'ha desenvolupat la síntesi de nous complexes de Coure(I), Coure(II) y Cu(III) utilitzant lligands macrocíclics de tipus triaza i hexaaza, i s'han estudiat la seves propietats estructurals així com la seva reactivitat. La reacció dels lligands triazacíclics H32m, H2Me33m i H33m amb sals de coure(II) dóna lloc a una reacció de desproporció de Cu(II) per obtenir-se en quantitats equimolars un complex organometàl·lic de Cu(III) i un complex de Cu(I). La caracterizació estructural exhaustiva dels complexes del tipus aryl-Cu(III) evidencia la formació d'un enllaç organometàl·lic entre l'àtom de Cu(III) i el carboni més próxim de l'anell aromàtic del lligand. Aquesta reacció, a més de representar una nova forma de desproporció en la química del Cu, suposa l'activació d'un enllaç C-H aromàtic a temperatura ambient que, mitjançant l'estudi cinètic d'aquesta desproporció per espectroscòpia UV-Vis, dels càlcul de l'efecte cinètic isotòpic utilitzant el lligand deuterat en el C-H de l'anell, juntament amb el recolzament teòrics dels càlculs DFT per a la optimització de geometries d'intermedis de reacció, ens permeten proposar un mecanisme de reacció pel nostre sistema, on l'activació de l'enllaç C-H aromàtic transcorre per la formació d'un enllaç de tipus agòstic C-H ? Cu(II),5 seguit de la desprotonació del C-H aromàtic per acció d'una base i posterior transferència electrònica per obtenir el complex organometàlic de Cu(III) i el complex de de Cu(I). En quant a la reactivitat d'aquests complexes organometàl·lics aryl-Cu(III) s'ha observat que una base en medi aquós causa la inestabilitat d'aquests compostos, evolucionant cap a la inserció d'un àtom d'oxigen sobre la posició activada de l'anell aromàtic, per a donar lloc a un complex dinuclear de Cu(II) amb dos grups fenoxo actuant de pont entre els àtoms metàl·lics. La reacció transcorre per un intermedi colorejat, caracteritzat com el complex ayl-Cu(III) monodesprotonat en una de les seves amines benzíliques, els quals s'observen igualment en la reacció dels correponents complexos de Cu(I) amb oxigen molecular (O2). És en els nostres sistemes en els quals es descriu per primera vegada la participació d'intermedis organometàl·lics Cu(III)-C en processos d'hidroxilació aromàtica, tals com el desenvolupat per l'enzim tirosinasa o per alguns dels seus models químics de síntesi.6,7,8 S'han estudiat les propietats magnètiques dels quatre bis(fenoxo)complexes de Cu(II) descrits, obtenint-se uns acoplaments de tipus antiferromagnètic o ferromagnètic de diversa magnitud, depenent del solapament orbitalari a l'enllaç Cu-O, a través del qual es produeix el superintercanvi. Nous complexos de Cu(I) sintetitzats amb lligands hexaazamacrocíclics han estat estudiats, i posant especial èmfasi a la seva reactivitat respecta a l'activació d'oxigen molecular (O2). S'ha observat una reactivitat diferenciada segons la concentració de complex de Cu(I) utilitzada, de manera que a altes concentracions s'obté un carbonato complex tetranuclear de Cu(II) per fixació de CO2 atmosfèric, mentre que a baixes concentracions s'observa la hidroxilació aromàtica intramolecular d'un dels anells benzílics del lligand, reacció que presumiblement transcorre per atac electrofílic d'un peroxo complex intermedi sobre el sistema ? de l'anell.6 Els resultats obtinguts en aquest treball ens mostren la facilitat per activar enllaços C-H aromàtics per metalls de transició de la primera sèrie (Cu, Ni) quan aquests estan suficientment pròxims a l'enllaç C-H, en unes condicions de reacció molt suaus (1atm., temperatura ambient). Els nous complexos organometàl·lics Aryl-Cu(III) són el producte d'una nova reacció de desproporció de Cu(II), així com un posició aromàtica activada que podria ser el punt de partida per l'estudi de funcionalització selectiva d'aquests grups aromàtics.

An in-situ surface electrochemistry approach toward whole-cell studies: Charge transfer between Geobacter sulfurreducens and electrified metal/electrolyte interfaces through linker molecules

Electrochemical reactivity and structure properties of electrogenic bacteria, Geobacter sulfurreducens (Gs) were studied to explore the heterogeneous electron transfer at the bacteria/electrode interface using electrochemical and in-situ spectroscopic techniques. The redox behavior of Gs adsorbed on a gold electrode, which is modified with a ω-functionalized self-assembled monolayer (SAM) of alkanethiols, depends strongly on the terminal group. The latter interacts directly with outermost cytochromes embedded into the outer membrane of the Gs cells. The redox potential of bacterial cells bound electrostatically to a carboxyl-terminated SAM is close to that observed for bacteria attached to a bare gold electrode, revealing a high electronic coupling at the cell/SAM interface. The redox potentials of bacterial cells adsorbed on amino- and pyridyl-terminated SAMs are significantly different suggesting that the outermost cytochromes changes their conformation upon adsorption on these SAMs. No redox activity of Gs was found with CH3-, N(CH3)3+- and OH-terminated SAMs. Complementary in-situ spectroscopic studies on bacteria/SAMs/Au electrode assemblies were carried out to monitor structure changes of the bacterial cells upon polarization. Spectro-electrochemical techniques revealed the electrochemical turnover of the oxidized and reduced states of outer membrane cytochromes (OMCs) in Gs, providing evidence that the OMCs are responsible for the direct electron transfer to metal electrodes, such as gold or silver, during the electricity production. Furthermore, we observed spectroscopic signatures of the native structure of the OMCs and no conformational change during the oxidation/reduction process of the microorganisms. These findings indicate that the carboxyl-anchoring group provides biocompatible conditions for the outermost cytochromes of the Gs, which facilitate the heterogeneous electron transfer at the microorganism/electrode interface.

An in situ surface electrochemistry approach towards whole-cell studies: the structure and reactivity of a Geobacter sulfurreducens submonolayer on electrified metal/electrolyte interfaces

A direct electron transfer process between bacterial cells of electrogenic species Geobacter sulfurreducens (Gs) and electrified electrode surfaces was studied to exploit the reactivity of Gs submonolayers on gold and silver surfaces. A submonolayer of Gs was prepared and studied to explore specifically the heterogeneous electron transfer properties at the bacteria/electrode interface. In situ microscopic techniques characterised the morphology of the Gs submonolayers under the operating conditions. In addition, complementary in situ spectroscopic techniques that allowed us to access in situ molecular information of the Gs with high surface selectivity and sensitivity were employed. The results provided clear evidence that the outermost cytochrome C in Gs is responsible for the heterogeneous electron transfer, which is in direct contact with the metal electrode. Feasibility of single cell in situ studies under operating conditions was demonstrated where the combination of surface-electrochemical tools at the nano- and micro-scale with microbiological approaches can offer unique opportunities for the emerging field of electro-microbiology to explore processes and interactions between microorganisms and electrical devices.

Probing the Electrocatalytic Oxygen Reduction Reaction Reactivity of Immobilized Multicopper Oxidase CueO

The bioelectrocatalytic (oxygen reduction reaction, ORR) properties of the multicopper oxidase CueO immobilized on gold electrodes were investigated. Macroscopic electrochemical techniques were combined with in situ scanning tunneling microscopy (STM) and surface-enhanced Raman spectroscopy at the ensemble and at the single-molecule level. Self-assembled monolayer of mercaptopropionic acid, cysteamine, and p-aminothiophenol were chosen as redox mediators. The highest ORR activity was observed for the protein attached to amino-terminated adlayers. In situ STM experiments revealed that the presence of oxygen causes distinct structure and electronic changes in the metallic centers of the enzyme, which determine the rate of intramolecular electron transfer and, consequently, affect the rate of electron tunneling through the protein. Complementary Raman spectroscopy experiments provided access for monitoring structural changes in the redox state of the type 1 copper center of the immobilized enzyme during the CueO-catalyzed oxygen reduction cycle. These results unequivocally demonstrate the existence of a direct electronic communication between the electrode substrate and the type 1 copper center.