Comparison and characterization of the [Fe4S4](2+/3+) centre in the wild-type and C77S mutated HiPIPs from Chromatium vinosum monitored by Mossbauer,Fe-57 ENDOR and EPR spectroscopies

Mossbauer. Fe-57 ENDOR, CW and pulsed EPR experiments were performed on the reduced and the oxidized high-potential iron proteins (HiPIPs) of the wild type (WT) and the C77S mutant from Chromatium vinosum. The EPR spectra of the oxidized WT and mutant show three species respectively having nearly th e same g-values but strongly changed spectral contributions. Relaxation tim es were estimated for oxidized WT and mutant at T=5 K with pulsed EPR. A-te nsor components of both iron pairs were obtained by Fe-57 ENDOR, proving a similar magnetic structure for the WT and the mutant. Electronic relaxation has to be taken into account at T=5 K in native and mutated oxidized HiPIP s to achieve agreement between Mossbauer and Fe-57 ENDOR spectroscopies. Th e Mossbauer spectroscopy shows that the oxidized cluster contains a pure fe rric and a mixed-valence iron pair coupled antiparallel. While all cluster irons from reduced C. vinosum WT are indistinguishable in the Mossbauer spe ctrum, the reduced C77S mutant shows a non-equivalence between the serine-b ound and the three cysteine-ligated iron ions. The Mossbauer parameters con firm a loss of the covalent character of the iron bond when S is replaced b y O and indicate a shift of the cluster's electron cloud towards the serine . Mossbauer spectra of the oxidized mutant can be simulated with two models : model I introduces a single electronic isomer with the serine always liga ted to a ferric iron. Model II assumes two equally populated electronic iso mers with the serine ligated to a ferric iron and a mixed-valence iron, res pectively. The latter model is in better agreement with EPR and NMR.