Conversion of fatty aldehydes to alka(e)nes and formate by a cyanobacterial aldehyde decarbonylase: cryptic redox by an unusual dimetal oxygenase

Abstract

Cyanobacterial aldehyde decarbonylase (AD) catalyzes conversion of fatty aldehydes (R-CHO) to alka(e)nes (R-H) and formate. Curiously, although this reaction appears to be redox-neutral and formally hydrolytic, AD has a ferritin-like protein architecture and a carboxylate-bridged dimetal cofactor that are both structurally similar to those found in di-iron oxidases and oxygenases. In addition, the in vitro activity of the AD from Nostoc punctiforme (Np) was shown to require a reducing system similar to the systems employed by these O(2)-utilizing di-iron enzymes. Here, we resolve this conundrum by showing that aldehyde cleavage by the Np AD also requires dioxygen and results in incorporation of (18)O from (18)O(2) into the formate product. AD thus oxygenates, without oxidizing, its substrate. We posit that (i) O(2) adds to the reduced cofactor to generate a metal-bound peroxide nucleophile that attacks the substrate carbonyl and initiates a radical scission of the C1-C2 bond, and (ii) the reducing system delivers two electrons during aldehyde cleavage, ensuring a redox-neutral outcome, and two additional electrons to return an oxidized form of the cofactor back to the reduced, O(2)-reactive form.

Figure 1

Reconstructed mass spectra illustrating the catalytic requirement for O₂ and the incorporation of ¹⁸O from ¹⁸O₂ into the formate product in the Np AD reaction. (A) Reactions in the continuous presence of O₂ (under air atmosphere, black bars), the absence of O₂ (gray), and the presence of O₂ during pre-incubation with the N/F/FR system (without substrate) but the absence of O₂ during the reaction (orange). (B) Reactions under an atmosphere of natural-abundance O₂ (red) or ¹⁸O₂ (99% isotopic purity; blue). (C) Control reactions in which the 2NPH derivative of either natural-abundance formate (green) or [¹³C]-formate (purple) was generated in H₂¹⁸O (70% enrichment) to quantify the extent of exchange of the oxygen atoms during the coupling reaction. The reactions in A were carried out at 21 °C for 20 h and contained, in a final volume of 0.40 mL, 0.10 mM Np AD, 0.5 mM R-¹³CHO substrate, 2 mM NADPH, and 100 μg/mL each of spinach ferredoxin and ferredoxin reductase in air-saturated 100 mM HEPES buffer, pH 7.4, containing 0.2% triton X-100. The reactions in B had the same composition but were carried out for 2 h.

Scheme 1. Two Alternative Explanations for the Similarity of Np AD to Di-iron Oxidases and Oxygenases and its Requirement for a Reducing System to Promote an Apparently Hydrolytic Reaction^a

^a(A) The reducing system provides n electrons (e⁻), and the most reduced form of the cofactor reacts with O₂ to generate the active state, which is more oxidized than the as-isolated form by 4 − n units (x + y − i − j = 4 − n); (B) O₂ is reduced in every cycle by four electrons from the reducing system.

Scheme 2

Hypothetical Mechanism for the Np AD Reaction