Enzymatic ω Oxidation

Abstract

Abstract Fatty acid and alkane hydroxylation in Pseudomonas oleovorans requires three protein components: DPNH-rubredoxin reductase, rubredoxin, and the ω-hydroxylase. Homogeneous preparations of the reductase catalyze electron transfer not only to the rubredoxin of P. oleovorans but also to the lower molecular weight rubredoxins of the anaerobic bacteria, Peptostreptococcus elsdenii, Clostridium pasteurianum, and Desulfovibrio gigas. In contrast, the reductase is inactive with nonheme iron proteins such as spinach ferredoxin, putidaredoxin, and adrenodoxin, all of which contain labile sulfide. The reductase transfers electrons directly to ferricyanide and dichlorophenolindophenol, and indirectly to cytochrome c; the latter reaction is completely dependent upon the presence of rubredoxin. DPNH is highly superior to TPNH as an electron donor. Since two electron equivalents are accepted per mole of reductase, as shown by anaerobic titrations with DPNH or dithionite, and since 1 mole of FAD is present, it is concluded that no active oxidation-reduction group is present other than the flavin. The absorbance noted at long wave lengths when the enzyme is quantitatively reduced by DPNH or by EDTA and light in the presence of DPN, but not when it is reduced by dithionite or by EDTA and light in the absence of DPN, suggests the formation of a charge transfer complex between the reduced flavoprotein and DPN. A stable semiquinone was not formed during the titrations, as judged by the spectral changes observed. An interaction between rubredoxin and the reductase was demonstrated both by spectrophotometric and fluorimetric techniques. Measurements of the decrease in fluorescence of the reductase in the presence of rubredoxin indicated that these components form a complex in a 1:1 ratio; the dissociation constant of the complex was determined to be 2.1 x 10-7 m. The energy of activation of the enzyme-catalyzed reduction of rubredoxin by DPNH was found to be 5400 cal per mole.