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Title: The enzyme mechanism of copper-containing nitrite reductase from Alcaligenes faecalis and its application in biosensor-like devices
Author: Krzeminski, Lukasz
ISNI:       0000 0004 2745 5056
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2012
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Copper-containing nitrite reductases (NiRs) are enzymes that efficiently reduce nitrite to nitric oxide in potent denitrifying bacteria. There has been an interest in their application in amperometric biosensors for monitoring nitrite levels in natural and waste waters. NiRs have a complex enzyme mechanism and depend on nitrite concentration and pH. Although the mechanism has been intensively studied, it is still controversial. In this thesis, a combined fluorescence and electrochemical method is used to simultaneously monitor the nitrite turn-over rate of a NiR from Alcaligenes faecalis S-6 and the oxidation state of the type-1 copper electron transfer site inside the enzyme. The catalytic activity of NiR is measured electrochemically by exploiting a direct electron transfer to fluorescently labelled enzyme molecules immobilised on modified gold, whereas the redox state of the type-1 copper site is determined from fluorescence intensity changes caused by F6rster resonance energy transfer (FRET) between a fluorophore attached to NiR and its type-1 copper site. Here, a determining role of internal electron transfer is found in NiR's mechanism. Moreover, the heterogeneous interfacial electron transfer to adsorbed NiRs is observed. The electro- activity and binding of labelled and unlabelled NiRs on gold modified with different self-assembled monolayers (SAMs) are studied to understand the effect of NiR labelling on the protein-electrode interactions. Here, electro-active and well-ordered biofilms of NiRs are found on electrodes with SAMs carrying the positive charge (negative NiRs) or when NiRs are modified with fluorophores that help orienting them on SAM-modified gold. Attempts have also been made to implement fluorescently labelled NiRs into the biosensor-like device with a fluorescence output using gold electrodes modified with tethered lipid bilayers (tBLMs) or conducting polymers (CPs). In spite of being unsuccessful, these studies give a better understanding of potential-dependent polymer dynamics and a control over protein immobilisation on functionalised lipid bilayer platforms.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available