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

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.

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.

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.

Conformational change induced by electron transfer in a monolayer of cytochrome P450 reductase adsorbed at the Au(110)-phosphate buffer interface.

The reflection anisotropy spectroscopy profiles of a variant of cytochrome P450 reductase adsorbed at the Au(110)-phosphate buffer interface depend on the sequence of potentials applied to the Au(110) electrode. It is suggested that this dependence arises from changes in the orientation of the isoalloxazine ring structures in the protein with respect to the Au(110) surface. This offers a method of monitoring conformational change in this protein by measuring variations in the reflection anisotropy spectrum arising from changes in the redox potential.

Controlling the formation of a monolayer of cytochrome P450 reductase onto Au surfaces.

The conditions necessary for the formation of a monolayer and a bilayer of a mutated form (P499C) of human cytochrome P450 reductase on a Au(110)/electrolyte interface have been determined using a quartz crystal microbalance with dissipation, atomic force microscopy, and reflection anisotropy spectroscopy (RAS). The molecules adsorb through a Au-S linkage and, for the monolayer, adopt an ordered structure on the Au(110) substrate in which the optical axes of the dipoles contributing to the RAS signal are aligned roughly along the optical axes of the Au(110) substrate. Differences between the absorption spectrum of the molecules in a solution and the RAS profile of the adsorbed monolayer are attributed to surface order in the orientation of dipoles that contribute in the low energy region of the spectrum, a roughly vertical orientation on the surface of the long axes of the isoalloxazine rings and the lack of any preferred orientation in the molecular structure of the dipoles in the aromatic amino acids. Our studies establish an important proof of principle for immobilizing large biological macromolecules to gold surfaces. This opens up detailed studies of the dynamics of biological macromolecules by RAS, which have general applications in studies of biological redox chemistry that are coupled to protein dynamics.

Spectral signatures of the surface reconstructions of Au(110)/electrolyte interfaces.

It is demonstrated that the (1 × 1) structure and the (1 × 2) and (1 × 3) surface reconstructions that occur at Au(110)/electrolyte interfaces have unique optical fingerprints. The optical fingerprints are potential, pH and anion dependent and have potential for use in monitoring dynamic changes at this interface. We also observe a specific reflection anisotropy spectroscopy signature that may arise from anions adsorbed on the (1 × 1) structure of Au(110).