Ordered multilayers of cytochrome P450 reductase adsorbed at Au(110)/phosphate buffer interfaces

Abstract

Bilayers and multilayers of a mutated form of cytochrome P450 reductase, P499C, form ordered structures when adsorbed at Au(110)/phosphate buffer interfaces. Reflection anisotropy spectroscopy (RAS) indicates that the degree of order increases with the number of layers and that the optical dipoles that contribute to the RAS profiles observed from the bilayer lie in planes that are oriented vertical to the surface and along one of the principal axes of the Au(110) surface. The contribution of the Au(110) substrate and the protein to the RAS profiles of the combined systems show an opposite dependence on the potential applied to the Au(110) electrode. The RAS profile observed from an adsorbed multilayer is dominated by the contribution from the protein.

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

“…The potentials of the oxidized and reduced forms of the P499C variant have been determined [6]. The preparation of ordered monolayers of P499C at the Au(110)-phosphate buffer interface has been described in detail previously [5,6] as have the electrochemical cell, the potentiostat, and the RAS instrument [5,6,10,11]. The latter 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 reflection from the crystal.…”

“…As in the previous work [5,6,9] the solutions used were NaH 2 PO 4 and K 2 HPO 4 (BDH, Analar grade) prepared with Millipore ultrapure water (18 M cm) and made oxygen free by purging with argon prior to use.…”

“…The potential was then varied in the sequence −0.557 to 0.056 to −0.652 V and a RAS profile was taken at each potential. Adsorption at both potentials is known to lead to the formation of ordered monolayers in the Au(110) surface [5,6]. The redox potentials for the different redox states of P499C CPR were measured electrochemically by redox titration.…”

“…Each is populated to a different extent in one-to four-electron-reduced CPR, based on the known redox potentials of the FAD and FMN flavin cofactors [4]. In previous work it was demonstrated that a variant of the protein can be assembled as an ordered monolayer on a Au(110)-phosphate buffer interface in both its oxidized [5] and reduced [6] forms mimicking the anchoring of the protein in the membrane. It has been shown previously that the technique of reflection anisotropy spectroscopy (RAS) can be used to monitor changes in the orientation of adenine adsorbed at a Au(110)-phosphate buffer interface as the potential applied to the Au(110) electrode is varied [7,8].…”

“…This opens up the possibility of using RAS to monitor conformational change on a millisecond timescale in cytochrome P450 reductase resulting from the transfer of electrons. This paper is part of a long term study directed at this aim [5,6,9] and describes the detailed changes that take place in the RAS of cytochrome P450 reductase adsorbed at Au(110)-phosphate buffer interfaces as the potential applied to the Au(110) electrode is varied.…”