Spectroscopy and Electrochemistry of Cytochrome P450 BM3-Surfactant Film Assemblies

Andrew K. Udit
Katharine D. Hagen
Peter J. Goldman
Andrew Star
James M. Gillan
Harry B. Gray
Michael G. Hill

Abstract

We report analyses of electrochemical and spectroscopic measurements on cytochrome P450 BM3 (BM3) in didodecyldimethylammonium bromide (DDAB) surfactant films. Electronic absorption spectra of BM3−DDAB films on silica slides reveal the characteristic low-spin Fe^III heme absorption maximum at 418 nm. A prominent peak in the absorption spectrum of BM3 Fe^II−CO in a DDAB dispersion is at 448 nm; in spectra of aged samples, a shoulder at ∼ 420 nm is present. Infrared absorption spectra of the BM3 Fe^II−CO complex in DDAB dispersions feature a time-dependent shift of the carbonyl stretching frequency from 1950 to 2080 cm^-1. Voltammetry of BM3-DDAB films on graphite electrodes gave the following results: Fe^III/II E_1/2 at −260 mV (vs SCE), ∼ 300 mV positive of the value measured in solution; Δ S°_rc, ΔS°, and ΔH° values for water-ligated BM3 in DDAB are −98 J mol^-1 K^-1, −163 J mol^-1 K^-1, and −47 kJ mol^-1, respectively; values for the imidazole-ligated enzyme are −8 J mol^-1 K^-1, −73 J mol^-1 K^-1, and −21 kJ mol^-1. Taken together, the data suggest that BM3 adopts a compact conformation within DDAB that in turn strengthens hydrogen bonding interactions with the heme axial cysteine, producing a P420-like species with decreased electron density around the metal center.