Huxley, Lucinda (2011)
Ph.D. thesis, University of Birmingham.
Transhydrogenase is an enzyme located in the cytoplasmic membrane of bacteria or the inner membrane of animal mitochondria. Using the energy of the proton electrochemical gradient (Δp), transhydrogenase translocates protons across the membrane whilst undergoing its redox reaction, in which hydride ion equivalents are transferred from NADH to NADP+ producing NAD+ and NADPH. Transhydrogenase comprises three components; dI binds NA(H), dIII binds NADP(H) and dII spans the membrane. Transhydrogenase is thought to function by way of a binding-change mechanism, which involves “open” and “occluded” conformations of the enzyme. In the open conformation, nucleotides can readily bind and dissociate from the enzyme but the hydride transfer reaction is blocked. In the occluded conformation, hydride transfer is permitted but the binding and release of nucleotides is blocked. Hydride transfer and proton translocation are coupled. The coupling is not well understood due to the lack of structural information about the membrane-spanning dII component. However, it is believed to involve conformational changes of the enzyme, particularly the dII and dIII components, resulting in the switch between the open and occluded conformations. Enzyme assays and tryptophan fluorescence experiments using apo-dIII in complex with dI revealed two features: Firstly, the binding of NADP(H) to dIII is very slow and is probably limited by the conversion from the occluded to the open conformation. Since the switch between the occluded and open conformations is thought to be central in the coupling of hydride transfer and proton translocation, the results presented here give an insight into the binding-change mechanism of transhydrogenase. Secondly, NAD(H) is able to slowly bind into the NADP(H)-binding site of dIII (the “wrong” site). This brought into question the specificity of the dIII component of transhydrogenase for NADP(H). The significance and likelihood of NAD(H) binding to dIII in the intact enzyme in the living cell are discussed.
This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder.
Repository Staff Only: item control page