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.
| Identifer | oai:union.ndltd.org:unibo.it/oai:amsdottorato.cib.unibo.it:684 |
| Date | 03 July 2008 |
| Creators | Dezi, Manuela <1977> |
| Contributors | Venturoli, Giovanni |
| Publisher | Alma Mater Studiorum - Università di Bologna |
| Source Sets | Università di Bologna |
| Language | English |
| Detected Language | English |
| Type | Doctoral Thesis, PeerReviewed |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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