Abstract
The reflection anisotropy spectroscopy profiles of a variant of cytochrome P450 reductase adsorbed at the Au(110)-phosphate buffer interface depend on the sequence of potentials applied to the Au(110) electrode. It is suggested that this dependence arises from changes in the orientation of the isoalloxazine ring structures in the protein with respect to the Au(110) surface. This offers a method of monitoring conformational change in this protein by measuring variations in the reflection anisotropy spectrum arising from changes in the redox potential.
Highlights
Cytochrome P450 reductase (CPR) is an electron transfer flavoprotein that in living systems is anchored to a membrane [1] and that carries out its electron transfer function by large changes in the relative orientation of two structural parts of the protein: the flavin adenine dinucleotide (FAD)- and flavin mononucleotide (FMN)-binding domains [2,3]
This is an instrumental effect arising from slight differences of the order of minutes of arc in the alignment of the first polarizer [13]. These differences between the two experiments do not have a significant effect on the analysis that follows and were minimized by a scaling of the results obtained for the reflection anisotropy spectroscopy (RAS) profiles of the Au(110)–buffer interface in the two experiments
Components of the absorption spectrum of cytochrome P450 reductase, including two features associated with the isoalloxazine rings, have been identified in the RAS profiles of ordered monolayers of the protein adsorbed at Au(110)–buffer interfaces
Summary
Cytochrome P450 reductase (CPR) is an electron transfer flavoprotein that in living systems is anchored to a membrane [1] and that carries out its electron transfer function by large changes in the relative orientation of two structural parts of the protein: the flavin adenine dinucleotide (FAD)- and flavin mononucleotide (FMN)-binding domains [2,3]. 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
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