Biochemical Characterization of Lauric Acid ω-Hydroxylation by a CYP4A1/NADPH-Cytochrome P450 Reductase Fusion Protein

The binding and hydroxylation of lauric acid by a genetically engineered and expressed fusion protein comprised of an N-truncated form of rat CYP4A1 linked to an N-truncated form of rat NADPH cytochrome P450 oxidoreductase (OR) (constructed by Fisher et al., (1992) Proc. Natl. Acad. Sci. USA 89, 10817-10822) has been characterized biochemically and compared to that shown by purified reconstituted rat CYP4A1 and liver microsomes from clofibrate-induced rats. In all systems lauric acid induced a Type I cytochrome P450 difference spectrum with Ks values in agreement with prior literature (range 10-18 μM). When provided with NADPH and oxygen but no other proteins or lipid, the fusion protein (called f4A1) catalyzed ω-hydroxylation of lauric acid with apparent Km and Vm of 3-4 μM and 4-5 nmol product/min/nmol P450 irrespective of buffer concentration or cation (NaPi or KPi, 25-200 mM); comparable values for reconstituted CYP4A1 and microsomes from clofibrate-induced rats are 9 μM and 34 min−1 and 5 μM and 10 min−1, respectively. (ω-1)-Hydroxylation of lauric acid was barely detectable (ω/(ω-1) = 135) with f4A1 or with reconstituted CYP4A1, but it accounted for up to 50% of total products formed by microsomes from clofibrate-induced rats. When added to the f4A1 system, OR stimulated hydroxylation up to fivefold at a OR:f4A1 ratio of 5:1; additionally, (ω-1)hydroxylation was routinely observed as a minor process (<4% of total product) in this system. These effects were also independent of buffer concentration. In contrast addition of cytochrome b5 (b5) caused a small (25%) decrease in ω-hydroxylation, while added phospholipid had no effect. However, the combination of OR, b5, and lipid stimulated turnover approximately 10-fold compared to f4A1 alone, and 11-hydroxylauric acid was regularly formed as a minor (3-4% of total) product. These observations indicate that the fusion protein f4A1 is functionally equivalent to reconstituted CYP4A1 with respect to binding and hydroxylation of lauric acid and suggest that it can be used as an alternative to reconstituted systems for structure-function and mechanistic studies of fatty acid ω-hydroxylation.