Moessbauer and EPR Studies of Azotobacter vinelandii Ferredoxin I

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Abstract
Azotobacter vinelandii ferredoxin I (FdI) is a small protein that contains one Fe4S4 cluster and one Fe3S4 cluster. Previous studies of FdI have shown that the redox potential of the Fe3S4 cluster and the MCD and CD spectra of the reduced Fe3S4 cluster are pH-dependent. Using Mössbauer and EPR spectroscopy, we have studied FdI in different oxidation states and at different pH values. Here, we report the spin Hamiltonian parameters of the oxidized (S = 1/2) Fe3S4 cluster at pH 7.4 and the reduced (S = 2) Fe3S4 cluster at pH 6.0 and 8.5. The pH dependence observed by MCD is also evident in the Mössbauer spectra which show a change of the magnetic hyperfine tensor for one Fe site of the valence-delocalized pair. The Fe4S4 cluster is ligated by cysteines 20, 39, 42, and 45, but not by the adjacent cysteine 24. Treatment of FdI with 3 equiv of ferricyanide alters the Fe4S4 cluster, yielding a new species, [Fe4S4]'. The S = 1/2 EPR signal of [Fe4S4]' has previously been attributed to the formation of a cysteine disulfide radical from Cys24 and cluster sulfide. Here we show that the EPR signal is broadened by 57Fe, indicating that the electronic spin is significantly coupled to the cluster iron. Consistent with this, substantial magnetic hyperfine interactions are observed by Mössbauer spectroscopy. In addition, the average isomer shift of the four Fe sites is smaller for [Fe4S4]' than for [Fe4S4]2+, indicating that the oxidation is iron-based to at least some extent. Incubation of FdI with excess ferricyanide destroys the Fe4S4 cluster but leaves the Fe3S4 cluster intact. Our studies of (3Fe)FdI show that the S = 1/2 spin of the Fe3S4 cluster interacts with another paramagnet, presumably a radical generated at the site left vacant by the removal of the Fe4S4 cluster.

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