Abstract
Aldehyde-deformylating oxygenase (ADO) is a ferritin-like nonheme-diiron enzyme that catalyzes the last step in a pathway through which fatty acids are converted into hydrocarbons in cyanobacteria. ADO catalyzes conversion of a fatty aldehyde to the corresponding alk(a/e)ne and formate, consuming four electrons and one molecule of O2 per turnover and incorporating one atom from O2 into the formate coproduct. The source of the reducing equivalents in vivo has not been definitively established, but a cyanobacterial [2Fe-2S] ferredoxin (PetF), reduced by ferredoxin-NADP(+) reductase (FNR) using NADPH, has been implicated. We show that both the diferric form of Nostoc punctiforme ADO and its (putative) diferric-peroxyhemiacetal intermediate are reduced much more rapidly by Synechocystis sp. PCC6803 PetF than by the previously employed chemical reductant, 1-methoxy-5-methylphenazinium methyl sulfate. The yield of formate and alkane per reduced PetF approaches its theoretical upper limit when reduction of the intermediate is carried out in the presence of FNR. Reduction of the intermediate by either system leads to accumulation of a substrate-derived peroxyl radical as a result of off-pathway trapping of the C2-alkyl radical intermediate by excess O2, which consequently diminishes the yield of the hydrocarbon product. A sulfinyl radical located on residue Cys71 also accumulates with short-chain aldehydes. The detection of these radicals under turnover conditions provides the most direct evidence to date for a free-radical mechanism. Additionally, our results expose an inefficiency of the enzyme in processing its radical intermediate, presenting a target for optimization of bioprocesses exploiting this hydrocarbon-production pathway.
