The Fe-hydrogenase from Megasphaera elsdenii undergoes direct electron exchange with glassy carbon electrodes. Cyclic voltammetry defines the catalytic performance of the enzyme over a continuous but precisely defined range of potentials. In the presence of H, and protons the bias of the enzyme towards H, production is readily visualised. Variation of the response with pH indicates that protein ionisations with pK of approximately 6.7 and 8.3 regulate the catalytic activity. Possible origins for these observations in the chemistry of the H,-activating site are discussed. The mid-wave potential of the catalytic response, Emld, is defined as the catalytic operating potential of the enzyme. Under an atmosphere of hydrogen Emid = -421 2 10 mV, pH 7 with a variation of -21 ? 4 mV pH-', 22°C. Deviation of Elnid from the thermodynamic potential of the hydrogedproton couple reflects the enzyme's influence over the catalysed reaction. Em,,, is the reduction potential of the H,-activating centre (H-cluster) in the absence of kinetic bottle-necks at other steps in the reaction mechanism.Keywords: hydrogenase ; Megasphaera elsdenii; iron-sulfur cluster; electrochemistry. which hydrogenases are able to couple intramolecular electron transfer to enzyme turnover is complicated by the intrinsic nature of reaction (1). Redox titration and spectroscopic analysis should be interpreted with care since chemically reduced hydrogenase will turnover the protons provided by water. Mechanistic information is also difficult to extract from traditional assays of hydrogenase activity which couple the enzyme to redox mediators since turnover is critically dependent on pH and potential which vary continually in such experiments [7-91. Understanding the chemistry of hydrogenases is further complicated by the proton's capacity to modulate enzyme activity in addition to being a substrate. Hydrogenase-catalysed oxidation of dihydrogen proceeds through heterolytic cleavage generating a proton and a hydride [lo], thus, deprotonation of the activesite base may favour H, oxidation. Proton concentration may also regulate hydrogenase activity through the reduction potentials of the metal centres certain of which have been shown to be pH dependent in Ni-Fe hydrogenases [ l l , 121. The origin of this behaviour and its significance to the catalytic mechanism are not yet certain. In this respect it is interesting to note that reduction and oxidation of [3Fe-4S] clusters in ferredoxins is accompanied by proton uptake and release respectively [I3 -171. The protons have been proposed to reside on the cluster itself, suggesting that metal centres may participate directly in electron and proton relays.Direct electrochemical methods provide a powerful tool with which to deconvolute the chemistry of redox proteins 118-201. A precisely defined, but continually varying, potential is applied to the sample and the resulting current measured. As a consequence, electron transfer and associated chemical transformations are defined in the potential and time domains p...

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An electrochemical biosensor for the detection of genetically modified food components is presented. The biosensor was based on 21-mer single-stranded oligonucleotide (ssDNA probe) specific to either 35S promoter or nos terminator, which are frequently present in transgenic DNA cassettes. ssDNA probe was covalently attached by 5′-phosphate end to amino group of cysteamine self-assembled monolayer (SAM) on gold electrode surface with the use of activating reagents – water soluble 1-ethyl-3(3′-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxy-sulfosuccinimide (NHS). The hybridization reaction on the electrode surface was detected via methylene blue (MB) presenting higher affinity to ssDNA probe than to DNA duplex. The electrode modification procedure was optimized using 19-mer oligoG and oligoC nucleotides. The biosensor enabled distinction between DNA samples isolated from soybean RoundupReady® (RR soybean) and non-genetically modified soybean. The frequent introduction of investigated DNA sequences in other genetically modified organisms (GMOs) give a broad perspectives for analytical application of the biosensor.

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