Mossbauer studies of the formation and reactivity of a quasi-stable peroxo intermediate of stearoyl-acyl carrier protein Δ9-desaturase

Stearoyl-ACP Delta(9)-desaturase (Delta 9D) is a diiron enzyme that catalyzes 18:O-ACP desaturation. Each subunit of homodimeric resting Delta 9D contains a diferric cluster, while chemical reduction by 4e(-) produces a diferrous cluster in each subunit. Reaction of 4e(-)-reduced Delta 9D with 18:0-ACP and O-2 yields a blue chromophore (lambda(max) similar to 700 nm) that exhibits a vibrational spectrum indicative of a mu-1,2-peroxo complex; this species has been designated peroxo Delta 9D. In contrast to other enzymic peroxodiiron intermediates, peroxo Delta 9D is long-lived (t(1/2) similar to 30 min at 25 degrees C) and decays via an oxidase reaction without formation of either H2O2 or product (18:1-ACP). In this work, optical, transient kinetic, and Mossbauer techniques have been used to further investigate the origin and nature of this unusual peroxodiiron complex. Rapid mixing of 4e(-) Delta 9D with O-2-equilibrated 18:O-ACP produced peroxo Delta 9D as revealed by a temperature-dependent, pseudo-first-order absorption increase at 700 nm (k = 46 s(-1) at 6 degrees C). The Mossbauer spectrum of peroxo Delta 9D, accounting for 96% of the total iron, consists of two quadrupole doublets present in equal proportions: delta(1) = 0.68(1) mm/s, and Delta E-Q(1) = 1.90(2) mm/s; delta(2) = 0.64(1) mm/s, and Delta E-Q(2) = 1.06(2) mm/s. Decay of the 700 nm optical band (k = 0.004 min(-1) at 6 degrees C) correlates with the complete conversion of peroxo Delta 9D into a complex called peroxo-cycled Delta 9D, which exhibits two new doublets present in equal proportions: delta(1) = 0.57(2) mm/s, and Delta E-Q(1) = 1.91(3) mm/s; delta(2) = 0.52(2) mm/s, and Delta E-Q(2) = 1.41(3) mm/s. Thus, peroxo Delta 9D contains two asymmetric diferric clusters and reacts to yield peroxo-cycled Delta 9D, also containing two asymmetric diferric clusters that most probably represent a substrate complex state. The clusters of both peroxo Delta 9D and peroxo-cycled Delta 9D have a diamagnetic ground state. Because peroxo Delta 9D and peroxo-cycled Delta 9D are observed only in the presence of 18:O-ACP, substrate binding appears to have introduced asymmetry into the Delta 9D diiron clusters. In situ photolysis of peroxo Delta 9D at 4.2 K in the Mossbauer cryostat caused the release of O-2 and the reappearance of a diferrous Delta 9D . 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 Mossbauer spectrum of peroxo Delta 9D, showing that the released O-2 was effectively sequestered within the active site.