Effect of H bond removal and changes in the position of the iron-sulphur head domain on the spin-lattice relaxation properties of the [2Fe-2S]2+ Rieske cluster in cytochrome bc1

Abstract

Here, comparative electron spin-lattice relaxation studies of the 2Fe-2S iron-sulphur (Fe-S) cluster embedded in a large membrane protein complex-cytochrome bc1-are reported. Structural modifications of the local environment alone (mutations S158A and Y160W removing specific H bonds between Fe-S and amino acid side chains) or in combination with changes in global protein conformation (mutations/inhibitors changing the position of the Fe-S binding domain within the protein complex) resulted in different redox potentials as well as g-, g-strain and the relaxation rates (T1-1) for the Fe-S cluster. The relaxation rates for T < 25 K were measured directly by inversion recovery, while for T > 60 K they were deduced from simulation of continuous wave EPR spectra of the cluster using a model that included anisotropy of Lorentzian broadening. In all cases, the relaxation rate involved contributions from direct, second-order Raman and Orbach processes, each dominating over different temperature ranges. The analysis of T1-1 (T) over the range 5-120 K yielded the values of the Orbach energy (EOrb), Debye temperature D and Raman process efficiency CRam for each variant of the protein. As the Orbach energy was generally higher for mutants S158A and Y160W, compared to wild-type protein (WT), it is suggested that H bond removal influences the geometry leading to increased strength of antiferromagnetic coupling between two Fe ions of the cluster. While D was similar for all variants (∼107 K), the efficiency of the Raman process generally depends on the spin-orbit coupling that is lower for S158A and Y160W mutants, when compared to the WT. However, in several cases CRam did not only correlate with spin-orbit coupling but was also influenced by other factors-possibly the modification of protein rigidity and therefore the vibrational modes around the Fe-S cluster that change upon the movement of the iron-sulphur head domain.