An amperometric enzyme electrode was studied based on the wild-type protein trimethylamine dehydrogenase (TMADH), which catalyses the oxidative N-demethylation of trimethylamine to produce dimethylamine and formaldehyde. Ferrocene derivatives were investigated electrochemically, as free diffusing electron acceptors for recycling of the prosthetic groups of the immobilised enzyme. Ferricinium had the highest rates but, inhibited the enzyme, possibly as a result of a conformational change initiated at the Val-344 residue where it binds close to the 4Fe-4S cluster, interrupting the electron transfer between flavin mononucleotide (FMN) and 4Fe-4S by changing the redox potential of one or both of the prosthetic groups. (Dimethylamino)methylene ferrocene (DMAMFe) (k(s) = 0.93 x 10(5) M(-1) s(-1)) did not show inhibition and was used as a comparison for steady-state characterisation. The sensor response was studied over the pH range 6.0-1.0. Plots of kcat/KM revealed two ionisations with pKa values of 7.5 and 10. The pKa of 10 was attributed to the ionisation of the secondary amine in DMAMFe, whereas the pKa of 7.5 was thought to reflect the ionisations of the intramolecular electron pathway. A TMADH/DMAMFe amperometric enzyme electrode was successfully used for the determination of TMA in different fish samples (detection limit: 2 mg TMA-N per lOOg wet fish muscle). The obtained results compared well with a reference method based on picric acid.

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A rational design for the site-specific immobilization of the protein trimethylamine dehydrogenase (TMADH) to facilitate charge transfer between enzyme and an electrode is described. Protein engineering and site-specific chemical modification have been used to extend the electron pathway from the protein surface to redox mediators. The kinetics of TMADH mutants (V344C and Y442C) modified with the iodoacetamide and 4-iodoacetamido 1-naphthole (IAN) showed that modification at position 344 has a more profound influence on intra-and inter-molecular electron transport, and the catalytic parameters k cat and K M app became a function of chemical modification. Ferricenium ion was shown to act as an electron acceptor for both mutants, but as its site of interaction is the residue 344, it was rejected for wiring in favour of [Fe(5-NH 2 -phen) 3 ] 2+ , the latter showing similar very fast homogeneous electron exchange kinetics, ideal for 'wire' construction. The Y442C mutant was successfully immobilised on to an electrode surface which had been chemically modified with the redox polymer, poly-[Fe(5-NH 2 -phen) 3 ] 2+ . This design enabled direct electrical communication between the enzyme and electrode. Using a partly oxidized polymer to limit the supply of oxidised electron acceptor, gave evidence for transition from the fast "0/2-cycle" to the "1/3-cycle" for the TMADH.

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