We have employed electron-nuclear double resonance (ENDOR) spectroscopy to study the bridged siroheme-[Fe4S4] cluster that forms the catalytically active center of the oxidized hemoprotein subunit (SiR°) of Escherichia coli NADPH-sulfite reductase. The siroheme 57Fe hyperfine coupling (Az = 27.6 MHz, Ay = 26.8 MHz) is similar to that of other high-spin heme systems (A ≈ 27 MHz). Bonding parameters obtained from the 14N hyperfine coupling constants of the siroheme pyrrole nitrogens are consistent with a model of a nonplanar π system of reduced aromaticity. The absence of hyperfine coupling to the 14N of an axial ligand, such as is observed for the histidine 14N of metmyoglobin (Az = 11.55 MHz), rules out the possibility that imidazolate acts as the bridge between the siroheme and the [Fe4S4] cluster. Proton ENDOR of the deuterium-exchanged protein indicates that H20 does not function as a sixth axial ligand and suggests that the ferrisiroheme is five-coordinate. 57Fe ENDOR measurements confirm the results of Mossbauer spectroscopy for the [Fe4S4] cluster. They also disclose a slight anisotropy of the cluster 57Fe coupling that may be associated with the mechanism by which the siroheme and cluster spins are coupled.
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