Using trimethylamine dehydrogenase in an enzyme linked amperometric electrode part 2. Rational design engineering of a 'wired' mutant

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.