Unusual NMR, EPR, and Mössbauer Properties of Chromatium vinosum 2[4Fe-4S] Ferredoxin

• Published in: Biochemistry (Easton) 1999-05, Vol.38 (19), p.6335-6345
• Main Authors: Kyritsis, Panayotis, Kümmerle, Rainer, Huber, J. Gaspard, Gaillard, Jacques, Guigliarelli, Bruno, Popescu, Codrina, Münck, Eckard, Moulis, Jean-Marc
• Format: Article
• Language: English
• Subjects: Bacterial Proteins; Chromatium - chemistry; Cysteine - chemistry; Dithionite - chemistry; Electron Spin Resonance Spectroscopy; Electron Transport; Ferredoxins - chemistry; Ferredoxins - genetics; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Models, Molecular; Mutagenesis, Site-Directed; Oxidation-Reduction; Spectroscopy, Mossbauer
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi982894u

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 unambiguously assigned here through NMR, EPR, and Mössbauer spectroscopy in combination with site-directed mutagenesis. The [4Fe-4S]2+/1+ cluster (cluster II) whose coordination sphere includes a two-turn loop between cysteines 40 and 49 was reduced by dithionite with an E°‘ of −460 mV. Its S = 1/2 EPR signal was 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, and they were associated with the particular structural elements of CvFd, including the two-turn loop between two ligands and the C-terminal α-helix. Bulk reduction of cluster I (E°‘ = −660 mV) was not possible for spectroscopic studies, but the full reduction of the protein was achieved by replacing valine 13 with glycine due to an ≈60 mV positive shift of the potential. At low temperatures, the EPR spectrum of the fully reduced protein was typical of two interacting S = 1/2 [4Fe-4S]1+ centers, but because the electronic relaxation of cluster I is much slower than that of cluster II, the resolved signal of cluster I was observed at temperatures above 20 K. Contact-shifted NMR resonances of β-CH2 protons were detected in all combinations of redox states. These results establish that electron transfer reactions involving CvFd are quantitatively different from similar reactions in isopotential 2[4Fe-4S] ferredoxins. However, the reduced clusters of CvFd have electronic distributions that are similar to those of clusters coordinated by the CysIxxCysIIxxCysIII···CysIVP sequence motif found in other ferredoxins with different biochemical properties. In all these cases, the electron added to the oxidized clusters is mainly accommodated in the pair of iron ions coordinated by CysII and CysIV.