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

Abstract

Mössbauer, 57Fe 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 the same g-values but strongly changed spectral contributions. Relaxation times were estimated for oxidized WT and mutant at T = 5 K with pulsed EPR. A-tensor components of both iron pairs were obtained by 57Fe 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 HiPIPs to achieve agreement between Mössbauer and 57Fe ENDOR spectroscopies. The Mössbauer spectroscopy shows that the oxidized cluster contains a pure ferric and a mixed-valence iron pair coupled antiparallel. While all cluster irons from reduced C. vinosum WT are indistinguishable in the Mössbauer spectrum, the reduced C77S mutant shows a non-equivalence between the serine-bound and the three cysteine-ligated iron ions. The Mössbauer parameters confirm a loss of the covalent character of the iron bond when S is replaced by O and indicate a shift of the cluster's electron cloud towards the serine. Mössbauer spectra of the oxidized mutant can be simulated with two models: model I introduces a single electronic isomer with the serine always ligated to a ferric iron. Model II assumes two equally populated electronic isomers with the serine ligated to a ferric iron and a mixed-valence iron, respectively. The latter model is in better agreement with EPR and NMR.

Dynamics of wild-type HiPIPs: a Cys77Ser mutant and a partially unfolded HiPIP

“…Therefore, a phenomenological fit is sufficient. As in an earlier investigation, Mo¨ssbauer spectra of the reduced HiPIP C. vinosum Cys77Ser mutant were fitted by two quadrupole doublets with a ratio of 3:1, accounting for the three cysteine-and the one serine-ligated cluster iron ions [15]. Also the spectra of the oxidized mutant were evaluated in this way.…”

“…The oxidized WT and the Cys77Ser mutant HiPIPs from C. vinosum show a qualitatively similar behaviour. All fits were performed by varying only the Mo¨ssbauer parameters isomer shift, d, quadrupole splitting, DE Q , and relaxation rate, c. All the other parameters were taken from Dilg et al [14,15] and kept at fixed values for all temperatures (see Table 2). The isomer shift and the quadrupole splitting decreased slightly with temperature.…”

“…Another manifestation of protein dynamics is the paramagnetic relaxation in oxidized HiPIPs. It was proved that consideration of this effect is essential for a consistent interpretation of Mo¨ssbauer, 57 Fe ENDOR and pulsed EPR experiments, even at liquid helium temperatures [14,15].…”

“…The fourfold cysteine coordination of the cluster and the magnetic coupling scheme within the cluster is maintained under the Cys77Ser exchange. However, the electronic charge density is shifted towards the serinate iron and the isomeric distribution is changed remarkably by the mutation [4,10,15].…”

Site-specific oxidation of the [Fe4S4] cubanes in high-potential iron sulfur proteins as probed by EPR and orientation-selective proton ENDOR spectroscopy:Ectothiorhodospira halophila I versusRhodocyclus tenuis

“…Since then, paramagnetic NMR has, at room temperature, assigned two site isomers, the same ones as in E. halophila HiPIP I but in a ratio of 60 to 40% [17]. More recently, EPR and M6ssbauer investigations have indicated three different species [18]. Another recent EPR study has analyzed, at X-band frequencies, systematically by simulation including a strain model, all available EPR data of HiPIPs in terms of number and g-factors of the individual components starting with a detailed analysis of C. vinosum, for which four species were postulated.…”