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Electrochemical Studies of Electron Transfer Proteins and Electroactive Biofilms
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Orientador(es)
Resumo(s)
Bioelectrochemistry has been recognized as a very important technique to get
relevant thermodynamic and kinetic information on diverse complex biological
systems. From the determination of redox potentials of metallic centers in small
electron carrier proteins to the resolution of more complex mechanisms in highly
organised enzymes, and even whole bacteria systems, the application of dynamic
electrochemical techniques has proved to be a powerful tool that has allowed to get
deeper in the understanding of such systems.
In the present thesis electrochemical techniques were used to study diverse
biochemical systems. Different approaches have been used, namely the classic bulk
systems in which the reactive species are in solution and immobilised systems where
proteins are physically constrained at the electrode surface. Within these, also
alternative methods were used, as membrane electrodes and physical adsorption of
the biological material.
Several systems of different complexity were object of study. Simple Enzymes
Small non-hemic proteins, essentially related to electron transfer processes, with
iron-sulfur centers, as desulforedoxin and other related iron-sulfur proteins,
associated with oxidative stress protection, namely superoxide reductases, were
investigated by cyclic and square wave voltammetry; its redox potentials and pH
dependence were determined.
Complex Enzymes and Catalytic Systems
Nitrogen Cycle Enzymes
Enzymes taking part in the nitrogen cycle were studied, namely the periplasmic
nitrate reductase isolated from Desulfovibrio desulfuricans ATCC 27774 and nitric
oxide reductase purifed from Pseudomonas nautica 617. The first one, responsible the nitrate reduction to nitrite, is the only known monomeric nitrate reductase
biding an iron-sulfur center and a molybdopterin co-factor. In this work it was
possible, for the first time, to observe the individual metal centers voltammetric
features. The electrocatalytic activity was also evaluated. The second enzyme, nitric
oxide reductase, promotes the two electrons reduction of NO to N2O. In this unique
work it was possible to obtain data from a nitric oxide reductase resulting from direct
electron transfer assays, accomplished by cyclic voltammetry. The demonstration of
the catalytic activity towards the oxygen reduction was achieved and the reduction
and catalysis of NO was also observed by direct electrochemistry. The pH
dependence of the catalytic center redox process was evaluated and it was possible
to show that the immobilized enzyme retained its native properties.
Production and Consumption of Hydrogen
Hydrogenase (Hase), isolated from Desulfovibrio gigas (Dg), is a multicenter enzyme
that catalyses the interconversion between H2 and H+, and that is involved in the
dissimilatory sulphate reduction pathway. In this work, the direct electrochemistry of
the Dg Hase, in bulk solution and also immobilised by adsorption, in turnover and
non-turnover conditions was studied. For the first time the redox features of the
enzyme metallic centers in non-catalytic conditions and without the addition of any of
the known enzyme inhibitors were attained. Besides, it was possible to tune the
activation and inactivation of the enzyme by dynamic potential control.
Approach to in vivo systems
Finally, the electrochemical behaviour of biofilms formed from pure cultures of
sulphate reducing bacteria, namely Desulfovibrio desulfuricans ATCC 27774 was
studied. The response of the biofilm on the electrodes was evaluated by the ratio of
current obtained in the presence/absence of the biofilm and its stability in time. For
the first time it was possible to observe that pure culture biofilms of sulphate reducing
bacteria are electroactive.