| Abstract
| - The ferredoxin from Chromatium vinosum (CvFd) exhibits sequence and structure peculiarities.Its two Fe4S4(SCys)4 clusters have unusually low potential transitions that have been unambiguouslyassigned here through NMR, EPR, and Mössbauer spectroscopy in combination with site-directedmutagenesis. The [4Fe-4S]2+/1+ cluster (cluster II) whose coordination sphere includes a two-turn loopbetween cysteines 40 and 49 was reduced by dithionite with an E°‘ of −460 mV. Its S = 1/2 EPR signalwas fast relaxing and severely broadened by g-strain, and its Mössbauer spectra were broad and unresolved.These spectroscopic features were sensitive to small perturbations of the coordination environment, andthey were associated with the particular structural elements of CvFd, including the two-turn loop betweentwo ligands and the C-terminal α-helix. Bulk reduction of cluster I (E°‘ = −660 mV) was not possiblefor spectroscopic studies, but the full reduction of the protein was achieved by replacing valine 13 withglycine due to an ≈60 mV positive shift of the potential. At low temperatures, the EPR spectrum of thefully reduced protein was typical of two interacting S = 1/2 [4Fe-4S]1+ centers, but because the electronicrelaxation of cluster I is much slower than that of cluster II, the resolved signal of cluster I was observedat temperatures above 20 K. Contact-shifted NMR resonances of β-CH2 protons were detected in allcombinations of redox states. These results establish that electron transfer reactions involving CvFd arequantitatively different from similar reactions in isopotential 2[4Fe-4S] ferredoxins. However, the reducedclusters of CvFd have electronic distributions that are similar to those of clusters coordinated by theCysIxxCysIIxxCysIII···CysIVP sequence motif found in other ferredoxins with different biochemicalproperties. In all these cases, the electron added to the oxidized clusters is mainly accommodated in thepair of iron ions coordinated by CysII and CysIV.
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