The kinetic and spectral characterization of the E. coli-Expressed mammalian CYP4A7: Cytochrome b₅ effects vary with substrate

The CYP4A gene subfamily is composed of a number of genes that encode cytochromes P450 from various species, including human, which catalyze the hydroxylation of various saturated and unsaturated fatty acids, including arachidonic acid and prostaglandins. CYP4A7, a fatty acid metabolizing cytochrome P450 from rabbit kidney, was expressed in E. coli by adding the first 10 codons of CYP17α producing final yields of 20 nmol/L in order to perform detailed kinetic and spectral studies. CYP4A7 metabolized arachidonate, laurate, and myristate, with maximum turnover numbers of 152, 130, and 64.5 min^-1 and corresponding K_m values of 74.5, 27, and 16.7 μM, respectively, in the presence of cytochrome b₅. In the absence of cytochrome b₅, CYP4A7 metabolized laurate and myristate with turnover numbers of 27.4 and 33.6 min^-1 and corresponding K_m values of 3.9 and 33 μM, respectively. Arachidonate was not metabolized in the absence of cytochrome b₅. Saturation kinetics studies performed with heme-depleted cytochrome b₅ (apo cytochrome b₅) yielded turnover numbers of 118 and 74 min^-1 and K_m values of 74 and 25 μM with laurate and myristate, respectively, indicating that cytochrome b₅ is not involved in electron transfer but rather plays a conformational role. Laurate perturbation of the visible absorption spectrum of CYP4A7 allowed for determination of the spectral binding constant (K_s) in the absence and presence of cytochrome b₅ (13 and 43 μM, respectively). In stopped-flow kinetics experiments, the flavin reduction (∼90 s^-1) and heine reduction (∼9 s^-1) phases of the monooxygenase reaction of CYP4A7 were not altered by the presence of cytochrome b₅. Estimations of the rate of CPR (0.3 s^-1) or cytochrome b₅ (9.1 s^-1) binding with CYP4A7 were also determined.