We have employed electron-nuclear double resonance (ENDOR) spectroscopy to study the 57Fe hyperfine interactions in the bridged-siroheme [4Fe-4S] cluster that forms the catalytically active center of the two-electron-reduced hemoprotein subunit of Escherichia coli NADPH-sulfite reductase (SiR2-). Previous electron paramagnetic resonance (EPR) and Mossbauer studies have shown that this enzyme oxidation state can exist in three distinct spectroscopic forms: (1) a wg = 2.29” EPR species that predominates in unligated SiR2-, in which the siroheme Fe2+ is believed to be in an S = 1 state; (2) a “g = 4.88” type of EPR species that predominates in SiR2- in the presence of small amounts of guanidinium sulfate, in which the siroheme Fe2+ is in an S = 2 state; and (3) a classical “g = 1.94” type of EPR species that is seen in SiR2- ligated with CO, in which the siroheme Fe2+ is in an S = 0 state. In all three species, the cluster is in the [4Fe-4S]1+ state, and two distinct types of Fe site are seen in Mossbauer spectroscopy. ENDOR studies confirm the Mossbauer assignments for the cluster 57Fe in the g = 1.94 state, with A values of 37, 37, and 32 MHz for site I and ca. 19 MHz for site II. The hyperfine interactions are not too different on the g = 2.29 state, with site I Fe showing more anisotropic A values of 32, 24, and 20 MHz (site II was not detected). The 57Fe ENDOR spectra of the g = 4.88 species are markedly different, however, with 57Fe hyperfine interactions from three, and most probably four, distinct sites being detected; all but one exhibit unusually anisotropic couplings. This degree of resolution of iron sites in a [4Fe-4S]1+ cluster is unprecedented. We have examined the 57Fe hyperfine interactions with the spin-coupling models previously developed to describe the g values of the SiR2- forms. The unusual properties of the iron sites within the cluster of the g = 4.88 form of SiR2- are not readily interpreted as simple manifestations of weak spin coupling between S = 2 ferroheme and unperturbed cluster and may well reflect real physical and/or chemical differences between the cluster in the g = 4.88 state and in the well-characterized g = 1.94 state.
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