Unique properties of purified, Escherichia coli-expressed constitutive cytochrome P4504A5

Cytochromes P450 of the 4A family metabolize a variety of fatty acids, pros taglandins, and eicosanoids mainly at the terminal carbon (omega-hydroxylat ion) and, to a lesser extent, at the penultimate carbon [(omega-1)hydroxyla tion]. In the present study, cytochrome P4504A5 (4A5) has been successfully expressed in Escherichia coli, with an average yield of enzyme of similar to 80 nmol/liter of cells. Spectroscopic characterization of the purified e nzyme, using electron paramagnetic resonance and absolute and substrate-per turbed optical difference spectroscopy, showed that the heme of resting 4A5 is primarily low spin, but is converted primarily to high spin by substrat e binding. The k(cat) and K-m values for laurate omega-hydroxylation were 4 1 min(-1) and 8.5 mu M, respectively, in the absence of cytochrome b5, and 138 min(-1) and 38 mu M, respectively, in the presence of cytochrome b5. Hy droxylation of palmitate was dependent on the presence of cytochrome b5; K- cat and K-m values were 48 min(-1) and 122 mu M, respectively. Hydroxylatio n of arachidonic acid was barely detectable and was unchanged by the additi on of cytochrome b5. (C) 1999 Academic Press.