Proton Environment of Reduced Rieske Iron−Sulfur Cluster Probed by Two-Dimensional ESEEM Spectroscopy

Abstract

The proton environment of the reduced [2Fe-2S] cluster in the water-soluble head domain of the Rieske iron−sulfur protein (ISF) from the cytochrome bc₁ complex of Rhodobacter sphaeroides has been studied by orientation-selected X-band 2D ESEEM. The 2D spectra show multiple cross-peaks from protons, with considerable overlap. Samples in which ¹H₂O water was replaced by ²H₂O were used to determine which of the observed peaks belong to exchangeable protons, likely involved in hydrogen bonds in the neighborhood of the cluster. By correlating the cross-peaks from 2D spectra recorded at different parts of the EPR spectrum, lines from nine distinct proton signals were identified. Assignment of the proton signals was based on a point-dipole model for interaction with electrons of Fe(III) and Fe(II) ions, using the high-resolution structure of ISF from Rb. sphaeroides. Analysis of experimental and calculated tensors has led us to conclude that even 2D spectra do not completely resolve all contributions from nearby protons. Particularly, the seven resolved signals from nonexchangeable protons could be produced by at least 13 protons. The contributions from exchangeable protons were resolved by difference spectra (¹H₂O minus ²H₂O), and assigned to two groups of protons with distinct anisotropic hyperfine values. The largest measured coupling exceeded any calculated value. This discrepancy could result from limitations of the point dipole approximation in dealing with the distribution of spin density over the sulfur atoms of the cluster and the cysteine ligands, or from differences between the structure in solution and the crystallographic structure. The approach demonstrated here provides a paradigm for a wide range of studies in which hydrogen-bonding interactions with metallic centers has a crucial role in understanding the function.