Rapid Reduction of the Diferric-Peroxyhemiacetal Intermediate in Aldehyde-Deformylating Oxygenase by a Cyanobacterial Ferredoxin: Evidence for a Free-Radical Mechanism

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.

A growing family of O2 activating dinuclear iron enzymes with key catalytic diiron(III)-peroxo intermediates: Biological systems and chemical models

“…Recent stopped flow UV-visible and Mössbauer spectroscopic data support the formation of a peroxy-hemiacetal species [101,102]. Indeed, in these experiments, the formation of a peroxo-Fe III Fe III species was interrupted by the appearance of an absorption band at 450 nm, which was depending on the presence of a substrate.…”

The Enigmatic P450 Decarboxylase OleT Is Capable of, but Evolved To Frustrate, Oxygen Rebound Chemistry

“…38,39 Reactions using C 12 FA, which formed the most alcohol products, were performed with H 2 16 O 2 or H 2 18 O 2 as an oxidant in sealed vials, quenched, and analyzed by MS following trimethylsilylation (TMS). A comparison of the demethylated [ m – 15] fragment ions from the TMS-derivatized alcohols is shown in Figure 2.…”

Beyond ferryl-mediated hydroxylation: 40 years of the rebound mechanism and C–H activation

“…In another variation of the rebound spectrum of reactions, aldehyde deformylation by the diiron enzyme ADO produces alkanes in a process that can be seen as a hydrogen rebound from Fe–OH 2 [171]. These reactions have recently been reviewed by Bollinger and Krebs [172].…”