The influence of the structure of the Au(110) surface on the ordering of a monolayer of cytochrome P450 reductase at the Au(110)/phosphate buffer interface.

C I Smith,B Khara,P Weightman,J H Convery,N S Scrutton

doi:10.1002/pssb.201350063

Abstract

The reflection anisotropy spectra (RAS) observed initially from Au(110)/phosphate buffer interfaces at applied potentials of −0.652 and 0.056 V are very similar to the spectra observed from ordered Au(110) (1 × 3) and anion induced (1 × 1) surface structures respectively. These RAS profiles transform to a common profile after cycling the potential between these two values over 72 h indicating the formation of a less ordered surface. The RAS of a monolayer of a P499C variant of the human flavoprotein cytochrome P450 reductase adsorbed at 0.056 V at an ordered Au(110)/phosphate buffer interface is shown to arise from an ordered layer in which the optical dipole transitions are in a plane that is orientated roughly normal to the surface and parallel to either the [11̄0] or [001] axes of the Au(110) surface. The same result was found previously for adsorption of P499C on an ordered interface at −0.652 V. The adsorption of P499C at the disordered surface does not result in the formation of an ordered monolayer confirming that the molecular ordering is strongly influenced by both the local structure and the long range macroscopic order of the Au(110) surface.

Highlights

While there has been considerable progress in determining the structure of proteins using techniques like X-Ray diffraction, there has been much less progress in measuring the conformational changes that are the key to their function
Cytochrome P450 reductase is chosen for these studies because it is an electron transfer flavoprotein that in living systems is anchored to a membrane and which carries out its electron transfer function by large changes in the relative orientation of two structural parts of the protein: the FAD and FMN binding domains
It has been demonstrated that the characteristic reflection anisotropy spectroscopy (RAS) profiles observed initially at applied potentials of −0.652 and 0.056 V from the Au(110)/phosphate buffer interface prepared by flame annealing transform to a common profile after cycling the potential between these two values over 72 h

Summary

Introduction

While there has been considerable progress in determining the structure of proteins using techniques like X-Ray diffraction, there has been much less progress in measuring the conformational changes that are the key to their function. It is possible in principle to induce conformational change in the protein by varying the potential applied to the Au(110) electrode and monitoring the change in real time using RAS. Prior to such studies it is necessary to establish the symmetry of cytochrome P450 reductase adsorbed at Au(110)/phosphate buffer interfaces at different potentials. This precondition is completed in the research reported in this paper

Results

Discussion

Conclusion