Search

• Pig heart mitochondrial membrane fragments can be deposited on functionalized gold electrodes. • The activity of Complex I, Complex II and Complex III can be monitored electrochemically measuring the reducing current of the electron mediator CoQ 1 in the presence of their respective substrates using cyclic voltammetry. • Electrochemical detection of respiratory chain complexes in their native membrane reflected unexpected interactions between different complexes. • Mitochondrial-membrane modified electrodes could be used to develop high-throughput electrochemical sensors for screening respiratory chain protein activity in their native environment. In the last decades enormous advances have been made in characterizing the atomic and molecular structure of respiratory chain supercomplexes [ 1 ]. However, it still remains a challenge to stitch this refined spatial atomistic description with functional information provided by biochemical studies of isolated protein material. Development of functional assays that detect respiratory chain complexes in their native membrane environment contribute to address the open questions related to the role played by their association and interactions. We present a characterization assay in which a functionalized gold electrode is modified with mitochondrial membrane fragments that allows monitoring electrochemically the activity of different respiratory chain complexes immersed in the mitochondrial membrane. We measure the intensity of the reducing current of the electron mediator CoQ 1 at the electrode surface and its variation upon addition of the corresponding enzymatic substrates. The activities of Complex I, Complex II and Complex III were monitored by the way in which they reduce the current, reflecting the amount of quinone reduced by the complexes in the presence of their substrates. We detect that CoQ 1 H 2 produced by Complex I remains partially trapped within the membrane and is more easily oxidized by Complex III or the electrode than the quinone reduced by Complex II. [Display omitted] [ABSTRACT FROM AUTHOR]

• Hydrogenase and ATPase reconstituted in a biomimetic membrane over an electrode. • Electro-enzymatic ATP regeneration for phosphorylation of glucose is achieved. • Electro-enzymatic ATP regeneration for synthesis of NADP+ is achieved. Adenosine-5-triphosphate (ATP) is the main energy vector in biological systems, thus its regeneration is an important issue for the application of many enzymes of interest in biocatalysis and synthetic biology. We have developed an electroenzymatic ATP regeneration system consisting in a gold electrode modified with a floating phospholipid bilayer that allows coupling the catalytic activity of two membrane-bound enzymes: NiFeSe hydrogenase from Desulfovibrio vulgaris and F 1 F o -ATP synthase from Escherichia coli. Thus, H 2 is used as a fuel for producing ATP. This electro-enzymatic assembly is studied as ATP regeneration system of phosphorylation reactions catalysed by kinases, such as hexokinase and NAD+-kinase for respectively producing glucose-6-phosphate and NADP+. [ABSTRACT FROM AUTHOR]