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.

Keywords: adsorption; cytochrome P450 reductase; gold; reflection anisotropy spectroscopy.

Figure 1

Schematic showing the oxidised form of P499C CPR adsorbed on the Au(110) surface. The FMN-binding domain is shown in light grey, the connecting domain in black and the FAD/NADP-domain in dark grey. The FAD and FMN cofactors are shown as sticks. It is important to note that the molecules are not isolated on the surface but are arranged in an ordered monolayer. The molecules are attached to the Au(110) surface through a Au–S bond. The schematic protein structure is derived from that of rat cytochrome P450 reductase [5]. The protein data accession code is 1AMO.

Figure 2

(a) RA spectra of Au(110) at −0.652 V (black line) and 0.056 V (grey line) before stepping the potential applied to the Au(110) surface. The line shows the energy at which the RAS signal was monitored as the applied potential was switched (Fig. 3). (b) RA spectra of Au(110) (grey line) and of a monolayer of P499C adsorbed on Au(110) (△) at 0.056 V. The subtraction of these spectra is shown by (◯). All spectra recorded in 0.1 M NaH₂PO₄/K₂HPO₄ at pH 7.2. It is important to note that the RAS profiles shown in (a) correspond to the same zero on the vertical scale since they were obtained in subsequent experiments. The same is true for the two profiles shown in (b).

Figure 3

Change in RA intensity as a function of time recorded at 2.7 eV while the potential is switched from −0.652 to +0.056 to −0.652 V every 300 s, first run (black line) and the last run after 72 h (grey) are plotted on the primary y-axis with the corresponding reflectivity on the secondary y-axis on the right of the figure. All the runs are recorded in 0.1 M NaH₂PO₄/K₂HPO₄ at pH 7.2.

Figure 4

RA spectra of Au(110) at −0.652 V (black line) and at 0.056 V (grey line) after stepping between −0.652 and 0.056 V for 72 h recorded in 0.1 M NaH₂PO₄/K₂HPO₄ at pH 7.2.

Figure 5

RA spectra of Au(110) at −0.652 V after stepping (black line) and after the addition of P499C CPR at −0.652 V (◯).

Figure 6

RA spectra of a monolayer of P499C CPR adsorbed on Au(110) at 0.056 V versus SCE at 45° (grey line), 55° (▲), 65° (◯), 75°(◆), 85° (+), 90° (×), 95°(□), 105° (◇), 115° (●), 125° (△) and 135° (black line) all recorded in 0.1 M NaH₂PO₄/K₂HPO₄ at pH 7.2.