title: 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.
authors.affiliation: Department of Physics, Oliver Lodge Laboratory, University of Liverpool, Liverpool L69 7ZE, United Kingdom.
authors.forename: J H
authors.initials: JH
authors.lastname: Convery
authors.forename: C I
authors.initials: CI
authors.lastname: Smith
authors.forename: B
authors.initials: B
authors.lastname: Khara
authors.forename: N S
authors.initials: NS
authors.lastname: Scrutton
authors.forename: P
authors.initials: P
authors.lastname: Harrison
authors.forename: T
authors.initials: T
authors.lastname: Farrell
authors.forename: D S
authors.initials: DS
authors.lastname: Martin
authors.forename: P
authors.initials: P
authors.lastname: Weightman
mesh_terms: D000327:Adsorption; D003460:Crystallization / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry; D006046:Gold / Q000737:chemistry*; D008422:Materials Testing; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000648:ultrastructure; D011485:Protein Binding; D013057:Spectrum Analysis / Q000379:methods*; D013499:Surface Properties
chemical_list: D004800:Enzymes, Immobilized; D006046:Gold; D009251:NADPH-Ferrihemoprotein Reductase