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

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.

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

“…As in previous work [1][2][3][4][5][6][7] experiments were performed on a Au(110) single crystal of 99.999% purity in the form of a disk of diameter 10 mm and 2 mm thick with an exposed surface area of 0.5 cm 2 . The orientation of the crystal by x-ray diffraction, its preparation by polishing and flame annealing, and insertion into the electrochemical cell have been described previously [1,2,[5][6][7].…”

“…The orientation of the crystal by x-ray diffraction, its preparation by polishing and flame annealing, and insertion into the electrochemical cell have been described previously [1,2,[5][6][7]. The electrochemical cell, potentiostat, referencing of potentials to a saturated calomel electrode (SCE), and the RAS instrument have also been described previously [1][2][3][4][5][6][7]12,13]. The RAS instrument yields a linear optical signal that is the difference in reflectivity from two orthogonal directions in the surface of plane polarized light at normal incidence and near normal reflection from the crystal.…”

“…This work is part of a long-term program aimed at developing the potential of the optical technique of reflection anisotropy spectroscopy (RAS) as a real time monitor of conformational changes in proteins adsorbed on surfaces [1][2][3][4]. There has been considerable progress in determining the structure of proteins using techniques such as x-ray diffraction but, due to the dearth of experimental techniques, less progress in measuring the conformational changes that are the key to their function.…”

“…In earlier work we have established the conditions under which ordered monolayers of CPR are adsorbed at Au(110)phosphate buffer interfaces [2][3][4]. The experiments employed an engineered form of CPR with a cysteine residue on the solvent accessible surface at the location of the Pro-499 residue (P499C) as described previously [2].…”

“…The cysteine residue * Author to whom correspondence should be addressed: peterw@liverpool.ac.uk facilitated the adsorption through the formation of a Au-S bond. It was found, using RAS, that monolayers adsorbed on Au(110) at applied potentials of −0.652 and 0.056 V, which are, respectively, the potentials at which P499C is fully reduced or oxidized in solution [2,4], were orientated with the optical transitions that give rise to the RAS profile lying in a plane that is orientated roughly normal to the surface and parallel to either the [110] or [001] axes of the Au(110) surface. It is clear that the long-range order of the Au(110) surfaces at the two applied potentials plays an important role in inducing the formation of ordered monolayers of adsorbed P499C [4].…”