Abstract
NADPH-cytochrome P450 reductase is a multi-domain redox enzyme which is a key component of the P450 mono-oxygenase drug-metabolizing system. We report studies of the conformational equilibrium of this enzyme using small-angle neutron scattering, under conditions where we are able to control the redox state of the enzyme precisely. Different redox states have a profound effect on domain orientation in the enzyme and we analyse the data in terms of a two-state equilibrium between compact and extended conformations. The effects of ionic strength show that the presence of a greater proportion of the extended form leads to an enhanced ability to transfer electrons to cytochrome c. Domain motion is intrinsically linked to the functionality of the enzyme, and we can define the position of the conformational equilibrium for individual steps in the catalytic cycle.
Highlights
The concept of an energy landscape for a folded protein requires that proteins exist as an equilibrium population of conformational states
cytochrome P450 reductase (CPR) is located on the endoplasmic reticulum where it is a key component of the P450 mono-oxygenase system which plays a central role in drug metabolism[18], and in the biosynthesis of secondary metabolites in plants[30]
There is evidence for the existence of domain motion in CPR and for its importance in catalysis[16, 18, 28, 37, 38], but it remains to be established precisely where in the reaction cycle it occurs. To address this question we examine the conformational equilibrium of CPR in solution using small-angle neutron scattering (SANS) and transient kinetics
Summary
The concept of an energy landscape for a folded protein requires that proteins exist as an equilibrium population of conformational states. An important family of ET proteins which depends on domain movement in this way is that of the diflavin reductases[16], which includes cytochrome P450 reductase (CPR; Fig. 1)[17,18,19], mammalian nitric oxide synthase (NOS)[20, 21], the cancer-related novel reductase 122, and methionine synthase reductase[23, 24], as well as the bacterial proteins sulfite reductase[25] and CYP BM326 These enzymes (or their reductase components) have three domains: an FMN-binding domain, related to flavodoxins, an FAD- and NADPH-binding domain, related to ferredoxin/ flavodoxin reductases, and a ‘linker’ domain, which may serve to position the other two domains. The power of solution scattering in studying domain organisation in proteins is well established; by using SANS rather than SAXS we are able to control the redox state of the enzyme precisely without the problems associated with reduction of the flavins by X-ray-induced photo-electrons[37] These experiments allow us to relate the domain movement to individual steps in the catalytic cycle of the enzyme
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
