Unusual NMR, EPR, and Mossbauer properties of Chromatium vinosum 2[4Fe-4S]ferredoxin

The ferredoxin from Chromatium vinosum (CvFd) exhibits sequence and structu re peculiarities. Its two Fe4S4(SCYs)(4) clusters have unusually low potent ial transitions that have been unambiguously assigned here through NMR, EPR , and Mossbauer spectroscopy in combination with site-directed mutagenesis. The [4Fe-4S](2+/1+) cluster (cluster II) whose coordination sphere include s a two-turn loop between cysteines 40 and 49 was reduced by dithionite wit h an E degrees' of -460 mV. Its S = 1/2 EPR signal was fast relaxing and se verely broadened by g-strain, and its Mossbauer spectra were broad and unre solved. These spectroscopic features were sensitive to small perturbations of the coordination environment, and they were associated with the particul ar structural elements of CvFd, including the two-turn loop between two lig ands and the C-terminal alpha-helix. Bulk reduction of cluster I (E degrees ' = -660 mV) was not possible for spectroscopic studies, but the full reduc tion of the protein was achieved by replacing valine 13 with glycine due to an approximate to 60 mV positive shift of the potential. At low temperatur es, the EPR spectrum of the fully reduced protein was typical of two intera cting S = 1/2 [4Fe-4S](1+) centers, but because the electronic relaxation o f 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 reso nances of beta-CH2 protons were detected in all. combinations of redox stat es. 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 dist ributions that are similar to those of clusters coordinated by the Cys(I)xx Cys(II)xxCys(III)...Cys(IV)P sequence motif found in other ferredoxins with different biochemical properties. In all these cases, the electron added t o the oxidized clusters is mainly accommodated in the pair of iron ions coo rdinated by Cys(II) and Cys(IV).