Electron Transfer Mechanism of the Rieske Protein from <i>Thermus thermophilus</i> from Solution Nuclear Magnetic Resonance Investigations

Abstract

We report nuclear magnetic resonance (NMR) data indicating that the Rieske protein from the cytochrome <i>bc</i> complex of <i>Thermus thermophilus</i> (<i>Tt</i>Rp) undergoes modest redox-state-dependent and ligand-dependent conformational changes. To test models concerning the mechanism by which <i>Tt</i>Rp transfers between different sites on the complex, we monitored ¹H, ¹⁵N, and ¹³C NMR signals as a function of the redox state and molar ratio of added ligand. Our studies of full-length <i>Tt</i>Rp were conducted in the presence of dodecyl phosphocholine micelles to solvate the membrane anchor of the protein and the hydrophobic tail of the ligand (hydroubiquinone). NMR data indicated that hydroubiquinone binds to <i>Tt</i>Rp and stabilizes an altered protein conformation. We utilized a truncated form of the Rieske protein lacking the membrane anchor (trunc-<i>Tt</i>Rp) to investigate redox-state-dependent conformational changes. Local chemical shift perturbations suggested possible conformational changes at prolyl residues. Detailed investigations showed that all observable prolyl residues of oxidized trunc-<i>Tt</i>Rp have <i>trans</i> peptide bond configurations but that two of these peptide bonds (Cys151–Pro152 and Gly169–Pro170 located near the iron–sulfur cluster) become <i>cis</i> in the reduced protein. Changes in the chemical shifts of backbone signals provided evidence of redox-state- and ligand-dependent conformational changes localized near the iron–sulfur cluster. These structural changes may alter interactions between the Rieske protein and the cytochrome <i>b</i> and <i>c</i> sites and provide part of the driving force for movement of the Rieske protein between these two sites