Mössbauer Studies of the Formation and Reactivity of a Quasi-Stable Peroxo Intermediate of Stearoyl-Acyl Carrier Protein Δ9-Desaturase

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Abstract
Stearoyl-ACP Δ9-desaturase (Δ9D) is a diiron enzyme that catalyzes 18:0-ACP desaturation. Each subunit of homodimeric resting Δ9D contains a diferric cluster, while chemical reduction by 4e- produces a diferrous cluster in each subunit. Reaction of 4e--reduced Δ9D with 18:0-ACP and O2 yields a blue chromophore (λmax ∼ 700 nm) that exhibits a vibrational spectrum indicative of a μ-1,2-peroxo complex; this species has been designated peroxoΔ9D. In contrast to other enzymic peroxodiiron intermediates, peroxoΔ9D is long-lived (t1/2 ∼ 30 min at 25 °C) and decays via an oxidase reaction without formation of either H2O2 or product (18:1-ACP). In this work, optical, transient kinetic, and Mössbauer techniques have been used to further investigate the origin and nature of this unusual peroxodiiron complex. Rapid mixing of 4e- Δ9D with O2-equilibrated 18:0-ACP produced peroxoΔ9D as revealed by a temperature-dependent, pseudo-first-order absorption increase at 700 nm (k = 46 s-1 at 6 °C). The Mössbauer spectrum of peroxoΔ9D, accounting for 96% of the total iron, consists of two quadrupole doublets present in equal proportions: δ(1) = 0.68(1) mm/s, and ΔEQ(1) = 1.90(2) mm/s; δ(2) = 0.64(1) mm/s, and ΔEQ(2) = 1.06(2) mm/s. Decay of the 700 nm optical band (k = 0.004 min-1 at 6 °C) correlates with the complete conversion of peroxoΔ9D into a complex called peroxo-cycled Δ9D, which exhibits two new doublets present in equal proportions: δ(1) = 0.57(2) mm/s, and ΔEQ(1) = 1.91(3) mm/s; δ(2) = 0.52(2) mm/s, and ΔEQ(2) = 1.41(3) mm/s. Thus, peroxoΔ9D contains two asymmetric diferric clusters and reacts to yield peroxo-cycled Δ9D, also containing two asymmetric diferric clusters that most probably represent a substrate complex state. The clusters of both peroxoΔ9D and peroxo-cycled Δ9D have a diamagnetic ground state. Because peroxoΔ9D and peroxo-cycled Δ9D are observed only in the presence of 18:0-ACP, substrate binding appears to have introduced asymmetry into the Δ9D diiron clusters. In situ photolysis of peroxoΔ9D at 4.2 K in the Mössbauer cryostat caused the release of O2 and the reappearance of a diferrous Δ9D·18:0-ACP complex with slightly changed parameters, suggesting a constrained cluster configuration was produced by the photolysis event. Annealing the photolyzed sample for 30 min at 77 K quantitatively restored the Mössbauer spectrum of peroxoΔ9D, showing that the released O2 was effectively sequestered within the active site.