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

Mossbauer spectra of the oxidized [Fe₄S₄]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 Mossbauer spectrum was simulated with two species contributing equally to the absorption area: two Fe³⁺ atoms couple to the 'ferric-ferric' pair, and one Fe²⁺ and one Fe³⁺ atom give the 'ferric- ferrous pair'. For the simulation of the Mossbauer 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 Mossbauer 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 Mossbauer 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 Mossbauer 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 Mossbauer spectrum, indicating a mixed-valence state of Fe²⁵⁺ for each.