Electrochemical Studies of Electron Transfer Proteins and Electroactive Biofilms

Abstract

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.