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

The temperature dependence of the mean square displacement of the (57)Fe nuclei due to motion faster than 100 ns are measured by temperature-dependent M?ssbauer spectroscopy for oxidized and reduced HiPIPs from Ectothiorhodospira halophila, Chromatium vinosum WT and a Cys77Ser mutant. The behaviour is interpretable in the frame of the general model of protein dynamics distinguishing two temperature intervals. The character of harmonic and quasi-diffusional modes in HiPIPs is discussed. Dynamic information obtained from M?ssbauer spectroscopy and Fe K-edge EXAFS are compared. Structure dynamics of the iron-sulfur cluster in the partially unfolded reduced HiPIP from C. vinosum was investigated by M?ssbauer spectroscopy and EXAFS, indicating an intact metal centre and a protein backbone with a largely collapsed secondary structure. The role of the cofactor during protein folding is discussed. Differences in the dynamics between the native protein and the molten globule are found at physiological temperatures only. The structure and dynamic behaviour of the [Fe(4)S(4)]Cys(3)Ser cluster in the Cys77Ser mutant of the HiPIP from C. vinosum are analysed. The temperature dependence of electron relaxation in oxidized HiPIPs is investigated by M?ssbauer spectroscopy and analysed theoretically, considering spin-spin and spin-lattice relaxation. The latter consists of contributions from direct phonon bottleneck and Orbach mechanisms. The data agree with former pulsed EPR results. Orbach relaxation is interpreted as due to transitions between electronic isomers of oxidized HiPIPs. With this interpretation, the energetic difference between both isomers equals the energy gap estimated from the temperature dependence of the Orbach relaxation.     

HIRA directly targets the enhancers of selected cardiac transcription factors during in vitro differentiation of mouse embryonic stem cells

Molecular Biology Reports - HIRA is a histone chaperone known to modulate gene expression through the deposition of H3.3. Conditional knockout of Hira in embryonic mouse hearts leads to...     

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

M?ssbauer, 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 M?ssbauer and 57Fe ENDOR spectroscopies. The M?ssbauer 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 M?ssbauer spectrum, the reduced C77S mutant shows a non-equivalence between the serine-bound and the three cysteine-ligated iron ions. The M?ssbauer 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. M?ssbauer 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.     

Simultaneous interpretation of Mössbauer, EPR and 57Fe ENDOR spectra of the [Fe4S4] cluster in the high-potential iron protein I Ectothiorhodospira halophila

4 S₄]^3 + and the reduced [Fe₄S₄]^2 + clusters in the high-potential iron protein I from Ectothiorhodospira halophila were measured in a temperature range from 5 K to 240 K. EPR measurements and ⁵⁷Fe electron-nuclear double resonance (ENDOR) experiments were carried out with the oxidized protein. In the oxidized state the cluster has a net spin S = 1/2 and is paramagnetic. As common in [Fe₄S₄]^3 + clusters, the M?ssbauer spectrum was simulated with two species contributing equally to the absorption area: two Fe^3 + atoms couple to the “ferric-ferric” pair, and one Fe^2 + and one Fe^3 + atom give the “ferric-ferrous pair”. For the simulation of the M?ssbauer spectrum, g-values were taken from EPR measurements. A-tensor components were determined by ⁵⁷Fe ENDOR experiments that turned out to be a necessary source of estimating parameters independently. In order to obtain a detailed agreement of M?ssbauer and ENDOR data, electronic relaxation has to be taken into account. Relaxing the symmetry condition in a way that the electric field gradient tensor does not coincide with g- and A-tensors yielded an even better agreement of experimental and theoretical M?ssbauer spectra. Spin-spin and spin-lattice relaxation times were estimated by pulsed EPR; the former turned out to be the dominating mechanism at T = 5 K. Relaxation times measured by pulsed EPR and obtained from the M?ssbauer fit were compared and yield nearly identical values. The reduced cluster has one additional electron and has a diamagnetic (S = 0) ground state. All the four irons are indistinguishable in the M?ssbauer spectrum, indicating a mixed-valence state of Fe^2.5 + for each. Received: 15 February 1999 / Accepted: 31 August 1999