Molecular assembly of electron trasferring flavoprotein complexes
thesisposted on 15.12.2014, 10:32 by Matthew Ronald Benjamin. Jones
The spectroscopic, kinetic and structural properties of electron transferring flavoprotein (ETF) from Methylophilus methylotrophus (sp. W3A1) have been investigated. Recombinant ETF has been expressed and purified, and its spectroscopic properties compared with those of native ETF by UV/visible spectrophotometry, fluorescence spectroscopy, circular dichroism spectroscopy and HPLC. The kinetics of electron transfer between W3A1 ETF and its physiological redox partner, trimethylamine dehydrogenase (TMADH), have been investigated in the presence and absence of glycerol to determine the effects of hydrodynamics on the electron transfer rate. It was found that the kinetics of electron transfer are unaffected by the concentrations of glycerol used in these analyses. Small angle X-ray solution scattering has been used to generate low resolution, model-independent molecular envelope structures for M. methylotrophus ETF, human ETF and Paracoccus denitrificans ETF. These analyses have indicated that all forms of ETF sample a range of conformations in solution. These studies suggest that an "induced fit" mechanism accounts for assembly of the TMADH-ETF electron transfer complex. Fluorescence and absorption spectroscopy studies of the TMADH-ETF complex have indicated that a series of conformational changes occur during complex assembly, and that electron transfer within mutant TMADH-ETF complexes can occur while the complex is in one or more metastable states. Furthermore, these studies have indicated that ETF undergoes a stable conformational change (termed structural imprinting) when it interacts transiently with TMADH. The imprinted form of ETF exhibits an enhanced rate of electron transfer to the artificial electron acceptor ferricenium. These studies show that the properties of electron transfer proteins can be affected lastingly by transient interaction with their physiological redox partners. This may have significant implications for our understanding of biological electron transfer reactions in vivo..