| Summary: | NADPH-cytochrome P450 reductase (CPR) is a multi-domain diflavin redox enzyme which is a key component of the P450 mono-oxygenase drug-metabolising system. This study was aimed to achieve an understanding of domain motions of human CPR which are believed to be intrinsic to its function and steps in its catalytic cycle. The protein and various mutants were prepared highly pure and catalytically active. Reduction of the oxidised form of the enzyme to its various possible redox states was accomplished using a variety of reducing agents. The redox states were characterised spectroscopically and stopped-flow techniques were used to further characterise the kinetics of the protein and mutants thereof under different conditions. Using small-angle X-ray scattering (SAXS) some evidence had been presented for a conformational equilibrium involving large-scale domain motions in this enzyme. The proposed equilibrium is studied using small-angle neutron scattering (SANS), under conditions where we are able to control the redox state of the enzyme precisely. It is shown that different redox states and buffer conditions have a profound effect on the conformational state of the enzyme and these findings are linked to kinetics studies. Different ways to model the data based on multi-state systems are presented. It is demonstrated that by altering the position of the conformational equilibrium by mutagenesis, the presence of a greater proportion of the extended form leads to an enhanced ability to transfer electrons to cytochrome c on the millisecond timescale. The position of the conformational equilibrium can therefore be defined for individual steps in the catalytic cycle. For the first time, the nature of the complex in solution between CPR and an electron transfer partner, cytochrome c, is described by means of deuteration and contrast matching in SANS. Finally, a time resolved structural study employing stopped-flow and SAXS measurements was designed and performed in an attempt to interpret the changes that CPR undergoes as a function of time.
|
|---|
