Conformational change in cytochrome P450 reductase adsorbed at a Au(110)—phosphate buffer interface induced by interaction with nicotinamide adenine dinucleotide phosphate

Abstract

Changes observed in the reflection anisotropy spectroscopy (RAS) profiles of monolayers of cytochrome P450 reductase adsorbed at Au(110)-electrolyte interfaces at 0.056 V following the addition of nicotinamide adenine dinucleotide phosphate (NADP(+)) are explained in terms of a simple model as arising from changes in the orientation of an isoalloxazine ring located in the flavin mononucleotide binding domain of the protein. The model also accounts for the changes observed in the RAS as the potential applied to the Au(110) surface is varied and suggests that differences in the dependence of the RAS profile of the adsorbed protein on the potential applied to the electrode in the absence and presence of NADP(+) are explicable as arising from a competition between the applied potential acting to reduce the protein and the NADP(+) to oxidize it.

Tripping the light fantastic in membrane redox biology: linking dynamic structures to function in ER electron transfer chains

“…To more fully understand the relationship between protein domain dynamics and CPR turnover, a description of conformational change during enzyme catalysis is required. In recent years, biophysical methods for studying dynamics relevant to catalysis have been developed for CPR and other members of the di‐flavin oxidoreductase family . In selected cases, these have been extended to identify trigger(s) (e.g.…”

“…Reflective anisotropy spectroscopy (RAS) is a ‘real‐time’ method that has been used to access the conformational landscape of CPR (Fig. A) . RAS is used to measure the difference in normal‐incidence reflectance for two different linear polarization directions , and has been used in recent years to monitor a conformational change in the number of complex redox enzymes that have been immobilized on gold electrodes .…”

“…A) . RAS is used to measure the difference in normal‐incidence reflectance for two different linear polarization directions , and has been used in recent years to monitor a conformational change in the number of complex redox enzymes that have been immobilized on gold electrodes . For CPR, an exposed cysteine residue was engineered on the surface of the enzyme (CPR variant P449C), and CPR‐Au(110) assemblies prepared that were suitable for combined electrochemical and reflectance anisotropy spectroscopy (RAS).…”

Real‐time analysis of conformational control in electron transfer reactions of human cytochrome P450 reductase with cytochrome c

“…Multiple spectroscopic techniques, including mass spectrometry , NMR , small‐angle X‐ray scattering , neutron scattering and reflection anisotropy , as well as construction of an ‘open’ yeast/human chimeric protein , have been used to demonstrate that CPR exists as a mixture of ‘open’ and ‘closed’ conformations, with long and short flavin–flavin distances, respectively. Moreover, pulsed electron–electron double resonance (PELDOR) spectroscopy measurements of two‐electron‐reduced (di‐semiquinone) forms of several diflavin oxidoreductases has shown that their conformational landscapes are ‘rugged’, with multiple conformers present .…”