The photosynthetic bacterial reaction center in native and artificial envirnoments: effects on light-induced electron transfer

In this thesis we focussed on the characterization of the reaction center (RC) protein purified from the photosynthetic bacterium Rhodobacter sphaeroides. In particular, we discussed the effects of native and artificial environment on the light-induced electron transfer processes. The native environment consist of the inner antenna LH1 complex that copurifies with the RC forming the so called core complex, and the lipid phase tightly associated with it. In parallel, we analyzed the role of saccharidic glassy matrices on the interplay between electron transfer processes and internal protein dynamics. As a different artificial matrix, we incorporated the RC protein in a layer-by-layer structure with a twofold aim: to check the behaviour of the protein in such an unusual environment and to test the response of the system to herbicides. By examining the RC in its native environment, we found that the light-induced charge separated state P+QB - is markedly stabilized (by about 40 meV) in the core complex as compared to the RC-only system over a physiological pH range. We also verified that, as compared to the average composition of the membrane, the core complex copurifies with a tightly bound lipid complement of about 90 phospholipid molecules per RC, which is strongly enriched in cardiolipin. In parallel, a large ubiquinone pool was found in association with the core complex, giving rise to a quinone concentration about ten times larger than the average one in the membrane. Moreover, this quinone pool is fully functional, i.e. it is promptly available at the QB site during multiple turnover excitation of the RC. The latter two observations suggest important heterogeneities and anisotropies in the native membranes which can in principle account for the stabilization of the charge separated state in the core complex. The thermodynamic and kinetic parameters obtained in the RC-LH1 complex are very close to those measured in intact membranes, indicating that the electron transfer properties of the RC in vivo are essentially determined by its local environment. The studies performed by incorporating the RC into saccharidic matrices evidenced the relevance of solvent-protein interactions and dynamical coupling in determining the kinetics of electron transfer processes. The usual approach when studying the interplay between internal motions and protein function consists in freezing the degrees of freedom of the protein at cryogenic temperature. We proved that the “trehalose approach” offers distinct advantages with respect to this traditional methodology. We showed, in fact, that the RC conformational dynamics, coupled to specific electron transfer processes, can be modulated by varying the hydration level of the trehalose matrix at room temperature, thus allowing to disentangle solvent from temperature effects. The comparison between different saccharidic matrices has revealed that the structural and dynamical protein-matrix coupling depends strongly upon the sugar. The analyses performed in RCs embedded in polyelectrolyte multilayers (PEM) structures have shown that the electron transfer from QA - to QB, a conformationally gated process extremely sensitive to the RC environment, can be strongly modulated by the hydration level of the matrix, confirming analogous results obtained for this electron transfer reaction in sugar matrices. We found that PEM-RCs are a very stable system, particularly suitable to study the thermodynamics and kinetics of herbicide binding to the QB site. These features make PEM-RC structures quite promising in the development of herbicide biosensors. The studies discussed in the present thesis have shown that, although the effects on electron transfer induced by the native and artificial environments tested are markedly different, they can be described on the basis of a common kinetic model which takes into account the static conformational heterogeneity of the RC and the interconversion between conformational substates. Interestingly, the same distribution of rate constants (i.e. a Gamma distribution function) can describe charge recombination processes in solutions of purified RC, in RC-LH1 complexes, in wet and dry RC-PEM structures and in glassy saccharidic matrices over a wide range of hydration levels. In conclusion, the results obtained for RCs in different physico-chemical environments emphasize the relevance of the structure/dynamics solvent/protein coupling in determining the energetics and the kinetics of electron transfer processes in a membrane protein complex.

Studio Sperimentale e Modellazione della Separazione di Proteine con Membrane di Affinità

Chromatography represents one of the most important and widely used unit operation in the biotechnology industry. However this technique suffers from several limitations such as high pressure drop, slow mass transfer through the diffusive pores and strong dependence of the binding capacity on flow rate. In this work, affinity membranes with improved capacity have been considered as an alternative technology for the capturing step in antibody manufacturing. Several affinity membranes have been prepared starting from various membrane supports. Different affinity ligands have been utilized like Protein A, the natural ligand of choice for antibodies, as well as synthetic ligands that exhibit affinity for the Fc portion of antibodies. The membranes have been characterized in detail: binding and elution performance was evaluated in adsorption experiments using pure IgG solutions, while membrane selectivity was evaluated using complex solutions like a cell culture supernatant. The most promising affinity membranes were extensively tested in dynamic experiments. The effects of operating parameters like feed concentration and flow rate on separation performances like binding capacity, selectivity and process yield have been studied in detail in order to find the optimal conditions for binding and elution steps. The membranes have been used over several complete chromatographic cycles to evaluate the effects of ageing and of membrane regeneration on dynamic binding capacity. A novel mathematical model is proposed that can describe all the chromatographic steps involved in the membrane affinity chromatography process for protein purification. The mathematical description is based on the species continuity equation coupled with a proper binding kinetic equation, and suitable to describe adequately the dispersion phenomena occurring both in the micro-porous membranes as well as in the extra-column devices used in the system. The model considers specifically all the different chromatographic steps, namely adsorption, washing and elution. The few relevant fitting parameters of the model were derived from a calibration with the experimental affinity cycles performed with pure IgG solutions, then the model is used to describe experimental data obtained in chromatographic cycles carried out with complex feeds as the cell culture supernatant. Simulations reveal a good agreement with experimental data in all the chromatography steps, both in the case of pure IgG solutions and for the cell culture supernatant considered.

Composti organostannici nell'ambiente marino e membrane biologiche: risposte molecolari e biochimiche nei molluschi bivalvi

Organotin compounds are worldwide diffused environmental contaminants, mainly as consequence of their extensive past use as biocides in antifouling paints. In spite of law restrictions, due to unwanted effects, organotin still persist in waters, being poorly degraded, easily resuspended from sediments and bioaccumulated in exposed organisms. The widespread toxicity and the possible threat to humans, likely to be organotin-exposed through contaminated seafood, make organotin interactions with biomolecules an intriguing biochemical topic, apart from a matter of ecotoxicological concern. Among organotins, tributyltin (TBT) is long known as the most dangerous and abundant chemical species in the Mediterranean Sea. Due to its amphiphilic nature, provided by three lipophilic arms and an electrophilic tin core, TBT can be easily incorporated in biomembranes and affect their functionality. Accordingly, it is known as a membrane-active toxicant and a mitochondrial poison. Up to now the molecular action modes of TBT are still partially unclear and poorly explored in bivalve mollusks, even if the latter play a not neglectable role in the marine trophic chain and efficiently accumulate organotins. The bivalve mollusk Mytilus galloprovincialis, selected for all experiments, is widely cultivated in the Mediterranean and currently used in ecotoxicological studies. Most work of this thesis was devoted to TBT effects on mussel mitochondria, but other possible targets of TBT were also considered. A great deal of literature points out TBT as endocrine disrupter and the masculinization of female marine gastropods, the so-called imposex, currently signals environmental organotin contamination. The hormonal status of TBT-exposed mussels and the possible interaction between hormones and contaminants in modulating microsomal hydroxilases, involved in steroid hormone and organotin detoxification, were the research topics in the period spent in Barcelona (Marco Polo fellowship). The variegated experimental approach, which consisted of two exposure experiments and in vitro tests, and the choice of selected tissues of M. galloprovincialis, the midgut gland for mitochondrial and microsomal preparations for subsequent laboratory assays and the gonads for the endocrine evaluations, aimed at drawing a clarifying pattern on the molecular mechanisms involved in organotin toxicity. TBT was promptly incorporated in midgut gland mitochondria of adult mussels exposed to 0.5 and 1.0 μg/L TBT, and partially degraded to DBT. TBT incorporation was accompanied by a decrease in the mitochondrial oligomycin-sensitive Mg-ATPase activity, while the coexistent oligomycin-insensitive fraction was unaffected. Mitochondrial fatty acids showed a clear rise in n-3 polyunsaturated fatty acids after 120 hr of TBT exposure, mainly referable to an increase in 22:6 level. TBT was also shown to inhibit the ATP hydrolytic activity of the mitochondrial F1FO complex in vitro and to promote an apparent loss of oligomycin sensitivity at higher than 1.0 μM concentration. The complex dose-dependent profile of the inhibition curve lead to the hypothesis of multiple TBT binding sites. At lower than 1.0 μM TBT concentrations the non competitive enzyme inhibition by TBT was ascribed to the non covalent binding of TBT to FO subunit. On the other hand the observed drop in oligomycin sensitivity at higher than 1.0 μM TBT could be related to the onset of covalent bonds involving thiolic groups on the enzyme structure, apparently reached only at high TBT levels. The mitochondrial respiratory complexes were in vitro affected by TBT, apart from the cytocrome c oxidase which was apparently refractory to the contaminant. The most striking inhibitory effect was shown on complex I, and ascribed to possible covalent bonds of TBT with –SH groups on the enzyme complexes. This mechanism, shouldered by the progressive decrease of free cystein residues in the presence of increasing TBT concentrations, suggests that the onset of covalent tin-sulphur bonds in distinct protein structures may constitute the molecular basis of widespread TBT effects on mitochondrial complexes. Energy production disturbances, in turn affecting energy consuming mechanisms, could be involved in other cellular changes. Mussels exposed to a wide range of TBT concentrations (20 - 200 and 2000 ng/L respectively) did not show any change in testosterone and estrogen levels in mature gonads. Most hormones were in the non-biologically active esterified form both in control and in TBT-treated mussels. Probably the endocrine status of sexually mature mussels could be refractory even to high TBT doses. In mussel digestive gland the high biological variability of microsomal 7-benzyloxy-4-trifluoromethylcoumarin-O-Debenzyloxylase (BFCOD) activity, taken as a measure of CYP3A-like efficiency, probably concealed any enzyme response to TBT exposure. On the other hand the TBT-driven enhancement of BFCOD activity in vitro was once again ascribed to covalent binding to thiol groups which, in this case, would stimulate the enzyme activity. In mussels from Barcelona harbour, a highly contaminated site, the enzyme showed a decreased affinity for the 7-benzyloxy-4-trifluoromethylcoumarin (BCF) substrate with respect to mussel sampled from Ebro Delta, a non-polluted marine site. Contaminant exposure may thus alter the kinetic features of enzymes involved in detoxification mechanisms. Contaminants and steroid hormones were clearly shown to mutually interact in the modulation of detoxification mechanisms. The xenoestrogen 17α-ethylenyl estradiol (EE2) displayed a non-competitive mixed inhibition of CYP3A-like activity by a preferential bond to the free enzyme both in Barcelona harbour and Ebro Delta mussels. The possible interaction with co-present contaminants in Barcelona harbour mussels apparently lessened the formation of the ternary complex enzyme-EE2-BCF. The whole of data confirms TBT as membrane toxicant in mussels as in other species and stresses TBT covalent binding to protein thiols as a widespread mechanism of membrane-bound-enzyme activity modulation by the contaminant.

Mitochondrial respiratory supercomplex association limits production of reactive oxygen species from Complex I

Evidence accumulated in the last ten years has demonstrated that a large proportion of the mitochondrial respiratory chain complexes in a variety of organisms is arranged in supramolecular assemblies called supercomplexes or respirasomes. Besides conferring a kinetic advantage (substrate channeling) and being required for the assembly and stability of Complex I, indirect considerations support the view that supercomplexes may also prevent excessive formation of reactive oxygen species (ROS) from the respiratory chain. Following this line of thought we have decided to directly investigate ROS production by Complex I under conditions in which the complex is arranged as a component of the supercomplex I1III2 or it is dissociated as an individual enzyme. The study has been addressed both in bovine heart mitochondrial membranes and in reconstituted proteoliposomes composed of complexes I and III in which the supramolecular organization of the respiratory assemblies is impaired by: (i) treatment either of bovine heart mitochondria or liposome-reconstituted supercomplex I-III with dodecyl maltoside; (ii) reconstitution of Complexes I and III at high phospholipids to protein ratio. The results of this investigation provide experimental evidence that the production of ROS is strongly increased in either model; supporting the view that disruption or prevention of the association between Complex I and Complex III by different means enhances the generation of superoxide from Complex I . This is the first demonstration that dissociation of the supercomplex I1III2 in the mitochondrial membrane is a cause of oxidative stress from Complex I. Previous work in our laboratory demonstrated that lipid peroxidation can dissociate the supramolecular assemblies; thus, here we confirm that preliminary conclusion that primary causes of oxidative stress may perpetuate reactive oxygen species (ROS) generation by a vicious circle involving supercomplex dissociation as a major determinant.