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

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.

Conformational change in cytochrome P450 reductase adsorbed at a Au(110)—phosphate buffer interface induced by interaction with nicotinamide adenine dinucleotide phosphate

“…As in previous work [1][2][3][4][5][6][7] experiments were performed on a Au(110) single crystal of 99.999% purity in the form of a disk of diameter 10 mm and 2 mm thick with an exposed surface area of 0.5 cm 2 . The orientation of the crystal by x-ray diffraction, its preparation by polishing and flame annealing, and insertion into the electrochemical cell have been described previously [1,2,[5][6][7].…”

“…The orientation of the crystal by x-ray diffraction, its preparation by polishing and flame annealing, and insertion into the electrochemical cell have been described previously [1,2,[5][6][7]. The electrochemical cell, potentiostat, referencing of potentials to a saturated calomel electrode (SCE), and the RAS instrument have also been described previously [1][2][3][4][5][6][7]12,13]. The RAS instrument 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 near normal reflection from the crystal.…”

“…As in the previous work [1][2][3][4][5][6][7], 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 CPR was engineered to have a cysteine residue at the location of the Pro-499 residue found in the wild-type enzyme as described previously [1,2], and the potentials of the oxidized form, 0.056 V, and the four successively reduced forms, −0.376, −0.465, −0.557, and −0.652 V, of the P499C variant have been determined [2].…”

“…This work is part of a long-term program aimed at developing the potential of the optical technique of reflection anisotropy spectroscopy (RAS) as a real time monitor of conformational changes in proteins adsorbed on surfaces [1][2][3][4]. There has been considerable progress in determining the structure of proteins using techniques such as x-ray diffraction but, due to the dearth of experimental techniques, less progress in measuring the conformational changes that are the key to their function.…”

“…In earlier work we have established the conditions under which ordered monolayers of CPR are adsorbed at Au(110)phosphate buffer interfaces [2][3][4]. The experiments employed an engineered form of CPR with a cysteine residue on the solvent accessible surface at the location of the Pro-499 residue (P499C) as described previously [2].…”