Propionate-3-nitronate oxidase from Penicillium atrovenetum is a flavoprotein which initiates the autoxidation of its substrate by O2.
On the fifth day following inoculation into an unstirred liquid surface culture, Penicillium atrovenetum abruptly, and reproducibly, secretes large quantities (2 g/liter) of the toxic antibiotic 3-nitropropionate into the medium. Concomitantly and with the same time course, crude extracts of the fungus acquire the ability to catalyze the oxidation of 3-nitropropionate by O2. We purified this activity some 300-fold to homogeneity and find it to be a soluble, dimeric (Mr = 73,000) flavoprotein oxidase having FMN as prosthetic group with lambda max = 363 and 433 nm. The preferred substrates are propionate-3-nitronate (3-NP-2) and O2 while the reaction products are malonate semialdehyde, NO2-, NO3-, O2-., and H2O2. Of 13 nitronates tested only butyrate-4-nitronate is more than 2% as reactive as 3-NP-2. 3-NP-2 (0.1 mM) rapidly reduces E-FMN anaerobically to E-FMNH., the flavin semiquinone (t1/2 less than 5 s), but reduces E-FMNH. to the fully reduced enzyme (E-FMNH2) very slowly (t1/2 approximately 900 s). The steady state turnover number with 0.1 mM 3-NP-2 and infinite O2 is 350 s-1. Therefore, the enzyme must oscillate almost exclusively between E-FMN and E-FMNH. during aerobic turnover. (Formula: see text). The complicated and non-integral reaction stoichiometry provides further support for this free radical mechanism. Each mole of 3-NP-. generated enzymatically initiates the nonenzymatic autoxidation of at least 2.2 mol of 3-NP-2 through a free radical chain reaction. An appropriate name for the newly characterized enzyme is propionate-3-nitronate oxidase.
Porter
D J
DJ
Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadephia 19104.
Bright
H J
HJ
D005486:Flavin Mononucleotide / Q000032:analysis; D005420:Flavoproteins / Q000302:isolation & purification; D005609:Free Radicals; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D009574:Nitro Compounds; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000032:analysis / Q000302:isolation & purification*; D010100:Oxygen / Q000378:metabolism*; D010407:Penicillium / Q000201:enzymology; D011422:Propionates / Q000378:metabolism*
D005420:Flavoproteins; D005609:Free Radicals; D009574:Nitro Compounds; D011422:Propionates; D005486:Flavin Mononucleotide; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; C053917:propionate-3-nitronate oxidase; C015392:3-nitropropionic acid; D010100:Oxygen