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

Abstract

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.

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

“…P499C thus has a cysteine group * Author to whom correspondence may be addressed: peterw@ liverpool.ac.uk on the solvent accessible surface of the protein which binds to the Au(110) surface [6]. The potentials of the oxidized and reduced forms of the P499C variant have been determined [6].…”

“…The experiments were conducted on a variant of the CPR protein, P499C, generated by site directed mutagenesis using a truncated form of the wild-type protein that lacks the helical N -terminal region that is required to anchor the protein on the endoplasmic reticulum [6]. P499C thus has a cysteine group * Author to whom correspondence may be addressed: peterw@ liverpool.ac.uk on the solvent accessible surface of the protein which binds to the Au(110) surface [6].…”

“…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.…”

Real-time reflectance-difference spectroscopy of GaAs molecular beam epitaxy homoepitaxial growth

“…An improved version of this spectrometer has been recently upgraded to 32 channels operation and applied to the study of the formation of organic films on Au (110) surfaces. 16 Recently, Hu et al 17 applied a multichannel RD spectrometer based on a rotating compensator as a polarization modulator to monitor, with a time resolution of 11.2 s, the growth of an organic film on a Cu (110) substrate.…”