Enzymatic ω-Oxidation
Abstract An inducible hydroxylase which catalyzes the conversion of fatty acids to their ω-hydroxy derivatives and n-alkanes to primary alcohols in the presence of molecular oxygen, a reduced pyridine nucleotide, rubredoxin, and a rubredoxin reductase has been solubilized and partially purified from Pseudomonas oleovorans. The bacterial reductase, which has higher activity with DPNH than with TPNH, may be replaced by highly purified ferredoxin reductase from spinach, for which TPNH is the preferred cofactor. The unlike an enzyme of similar function occurring in liver microsomes, contains no cytochrome P-450. The enzyme is of high molecular weight (estimated as about 2 x 106 by chromatography on a calibrated agarose column), but appears to dissociate into smaller units in the presence of dodecyl sulfate and mercaptoethanol. Iron was determined by emission spectroscopy to be present in the amount of 4.2 g atoms/2 x 106 g of protein, whereas only traces of FAD and heme were found. The electron paramagnetic resonance spectrum showed signals typical of rhombic iron (g = 4.3), a free radical (g = 2.003), and a labile sulfide-containing non-heme iron protein (g = 1.94), with a minor component at 2.03), but the significance of these resonances with respect to the catalytic function of the iron is not yet known. In the presence of rubredoxin, a rubredoxin reductase, and the ω-hydroxylase, the hydroxylation of octane was shown to yield n-octanol in equimolar amount to the amounts of TPNH and molecular oxygen consumed. This stoichiometry corresponds to that expected of a monooxygenase or mixed function oxidase, as follows: RCH3 + O2 + D(T)PNH + H+ → RCH2OH + D(T)PN+ + H2O. In addition to octane, which is the most active substrate, other n-alkanes from C6 to C14, cyclohexane, methylcyclohexane, dimethylhexane, fatty acids from C6 to C12, and certain glyceride derivatives of fatty acids all undergo hydroxylation as judged by the substrate-dependent disappearance of TPNH in the complete enzyme system. The ω-hydroxylase is extensively inhibited by cyanide and 8-hydroxyquinoline. Cyanide was shown to act as a noncompetitive inhibitor with respect to laurate and to have a Ki of 3.1 x 10-4m. The Km values of hexanoate, heptanoate, and nonanoate are 2.2 x 10-2m, 5.2 x 10-3m, and 6.9 x 10-4m, respectively.