Circular Dichroism and Magnetic Circular Dichroism Studies of the Reduced Binuclear Non-Heme Iron Site of Stearoyl-ACP Δ9-Desaturase:  Substrate Binding and Comparison to Ribonucleotide Reductase

Stearoyl-acyl carrier protein (stearoyl-ACP) Δ9-desaturase (Δ9D) catalyzes the insertion of a cis double bond between the 9 and 10 positions of the stearoyl-ACP to convert it to oleoyl-ACP. The binuclear non-heme iron active site of the fully reduced enzyme (reduced Δ9D) and its substrate-bound form (stearoyl-ACP Δ9D) have been studied using a combination of circular dichroism (CD) and magnetic circular dichroism (MCD) to probe their geometric and electronic structures. CD and MCD in the near-IR region probe the ligand-field d−d transitions of the ferrous sites. Variable-temperature variable-field (VTVH) MCD combined with a spin-Hamiltonian analysis including the zero-field splitting (ZFS) of both irons and the exchange coupling (J) between the irons due to bridging ligation is used to probe their ground-state properties. These ground- and excited-state results indicate that the active site of reduced Δ9D has two equivalent 5-coordinate irons in a distorted square pyramidal geometry. They are weakly antiferromagnetically coupled with large negative and equivalent ZFSs (D 1 = D 2 < 0), which gives a diamagnetic ground state interacting with low-lying paramagnetic excited states. Addition of substrate causes a significant change in both the excited states and the nature of the ground state. These spectral changes indicate that one of the irons becomes 4-coordinate, while the other distorts toward a trigonal bipyramidal geometry. The two irons remain weakly antiferromagnetically coupled. However, this geometry change modifies their ZFSs (D 1 < 0 and D 2 > 0), which results in a new ground state, M s = ±1, with a low-lying M s = ±2 first excited state. These results are the first direct evidence that the stearoyl-ACP binding strongly perturbs the active site, creating an additional open coordination position that correlates with enhanced dioxygen reactivity. These results are correlated with the X-ray crystal structure and compared to the related enzyme, ribonucleotide reductase, to gain insight into geometric and electronic structure contributions to dioxygen reactivity.