Substrate-triggered addition of dioxygen to the diferrous cofactor of aldehyde-deformylating oxygenase to form a diferric-peroxide intermediate

Abstract

Cyanobacterial aldehyde-deformylating oxygenases (ADOs) belong to the ferritin-like diiron-carboxylate superfamily of dioxygen-activating proteins. They catalyze conversion of saturated or monounsaturated C(n) fatty aldehydes to formate and the corresponding C(n-1) alkanes or alkenes, respectively. This unusual, apparently redox-neutral transformation actually requires four electrons per turnover to reduce the O2 cosubstrate to the oxidation state of water and incorporates one O-atom from O2 into the formate coproduct. We show here that the complex of the diiron(II/II) form of ADO from Nostoc punctiforme (Np) with an aldehyde substrate reacts with O2 to form a colored intermediate with spectroscopic properties suggestive of a Fe2(III/III) complex with a bound peroxide. Its Mössbauer spectra reveal that the intermediate possesses an antiferromagnetically (AF) coupled Fe2(III/III) center with resolved subsites. The intermediate is long-lived in the absence of a reducing system, decaying slowly (t(1/2) ~ 400 s at 5 °C) to produce a very modest yield of formate (<0.15 enzyme equivalents), but reacts rapidly with the fully reduced form of 1-methoxy-5-methylphenazinium methylsulfate ((MeO)PMS) to yield product, albeit at only ~50% of the maximum theoretical yield (owing to competition from one or more unproductive pathway). The results represent the most definitive evidence to date that ADO can use a diiron cofactor (rather than a homo- or heterodinuclear cluster involving another transition metal) and provide support for a mechanism involving attack on the carbonyl of the bound substrate by the reduced O2 moiety to form a Fe2(III/III)-peroxyhemiacetal complex, which undergoes reductive O-O-bond cleavage, leading to C1-C2 radical fragmentation and formation of the alk(a/e)ne and formate products.

Figure 1

Mössbauer spectra of Np ADO isolated from Ec grown in minimal medium with ⁵⁷Fe supplementation. (A) Spectrum of the aerobically isolated ADO in the presence of n-decanal recorded at 4.2 K and magnetic fields of 53 mT (vertical bars) and simulation of the experimental spectrum with parameters provided in the text (purple line). (B) Spectrum of the sample in A recorded with a magnetic field of 6 T (vertical bars) applied parallel to the γ beam and simulation thereof with parameters described in the text (purple line). (C) 4.2-K/53 mT spectrum of anaerobically isolated ADO in the presence of n-decanal (vertical bars); the 6%-contribution from the Fe₂^III/III complex in this sample is shown in purple.

Figure 2

SF-Abs experiment monitoring the reaction at 5 °C of Fe₂^II/II-ADO (0.35 mM) with O₂ (0.9 mM) (A) in the absence and (B) in the presence of 10 mM decanal. The arrows in B indicate the (dis)appearance of an intermediate absorbing at ~ 450 nm only in the presence of the aldehyde substrate.

Figure 3

Characteristics of the formation and decay of the 450-nm-absorbing complex. (A) Requirement for the aldehyde functional group for substrate triggering of intermediate formation in reaction of Fe₂^II/II-ADO with O₂. A solution of 200 μM Fe₂^II/II-ADO, 2 mM substrate or analog [n-nonanal (green), n-octanoic acid (red), n-nonan-1-ol (blue), n-nonane (purple), or no substrate (black)] was mixed in the SF-Abs instrument at 5 °C with O₂-saturated (1.8 mM) buffer (50 mM sodium Hepes, 10% glycerol, pH 7.5). The reaction was monitored at 450 nm with PDA detection. (B) Assessment of the intermediate's photolytic lability by SF-Abs spectroscopy monitored at 450 nm. A solution of 700 μM Fe₂^II/II-ADO and 10 mM n-decanal was mixed with an equal volume of an O₂-saturated buffer solution at 5 °C. ΔA_450nm-vs-time traces were recorded with PDA (green trace) or PMT (blue trace) detection. Regression analysis of the latter trace was carried out according to Equation 6, giving rate constants mentioned in the text. Control experiments carried out in the absence of n-decanal with PDA and PMT detection are shown as black and grey traces, respectively.

Figure 4

Characterization of the 450-nm-absorbing complex by FQ Mössbauer spectroscopy. (A) 4.2-K/53-mT Mössbauer spectrum of a FQ sample prepared by reacting Fe₂^II/II-ADO•1-[¹³C]-octanal with O₂-saturated buffer for 32 s (vertical bars). The contribution of unreacted Fe₂^II/II-ADO•1-[¹³C]-octanal (14%) and the Fe₂^III/III-ADO (6%) are shown as light blue and purple lines, respectively. (B) Removal of the contributions from the Fe₂^II/II-ADO•1-[¹³C]-octanal and Fe₂^III/III-ADO from A yields the spectrum of the intermediate (vertical bars), which can be simulated as two quadrupole doublets of equal intensity (blue and green shaded areas and red line) with parameters quoted in the text. (C) 4.2-K/6-T spectrum of the 32-s sample described above (vertical bars) and simulation of the sub spectra of the Fe₂^III/III-peroxide intermediate (blue and green shaded areas) as described in the text. The spectrum of the minor Fe₂^III/III contribution is shown in purple, and the summation of the contributions from the intermediate and Fe₂^III/III contaminant is shown as the red line plotted on top of the data.

Figure 5

Sequential-mixing SF-Abs experiments establishing the ability of the Fe₂^III/III-peroxide complex to oxidize ^MeOPMS. The Fe₂^II/II-ADO•1 [¹³C]-octanal reactant solution was mixed with O₂-saturated buffer, the solution was allowed to react for the delay time (t_delay) indicated in the figure, the aged reaction solution was then mixed with a solution of dithionite-reduced ^MeOPMS, and spectra were acquired following the second mix. (A) Selected spectra from the experiment with t_delay = 32 s, reflecting the rapid oxidation of reduced ^MeOPMS. (B) ΔA₃₈₈-vs-time traces for experiments with different delay times between the two mixes [t_delay = 0.01 s (blue), 32 s (green), 500 s (red)]. A control experiment, in which ADO was omitted is shown in black. Dashed lines are fits of Eq. 7 to the data.

Figure 6

Determination of the reductant:formate stoichiometry upon reduction of the Fe₂^III/III-peroxide complex with ^MeOPMS. The dotted fit line gives a formate:reductant ratio of ~ 0.5, which is half of the theoretical yield.

Figure 7

Mössbauer-spectroscopic characterization of the ADO cofactor following the productive decay of the Fe₂^III/III-peroxide intermediate that is initiated by reduced ^MeOPMS. (A) Experimental 4.2-K/53-mT spectrum after reaction of the pre-formed complex with dithionite-reduced ^MeOPMS for 0.56 s at 5 °C. (B) Difference spectrum (vertical bars) prepared by subtracting the spectrum of the sample quenched before reduction (Figure 4A) from spectrum A. In this presentation, the features associated with the Fe₂^III/III-peroxide complex point upwards (the pink line is the experimental “reference spectrum” shown in Figure 4B scaled to 46% intensity) and the features associated with the product of the productive decay point downwards. (C) “Reference spectrum” of the product of the reaction of the Fe₂^III/III-peroxide intermediate with reduced ^MeOPMS (vertical bars) and quadrupole doublet simulations with parameters quoted in the text (solid lines). (D) Spectrum of a sample prepared by allowing the Fe₂^III/III-peroxide intermediate to decay at 5 °C for 30 min in the absence of reduced ^MeOPMS.

Scheme 1

Proposed catalytic mechanism for the conversion of aldehyde to alk(a/e)ne and formate by ADO.^, The new hydrogen atom incorporated into the alk(a/e)ne product ultimately comes from solvent, but the proximal donor could be either a solvent ligand (green arrows) or an amino acid residue (depicted as H-X; purple arrows).