Breaking and re-forming the disulfide bond at the high-potential, respiratory-type Rieske [2Fe-2S] center of thermus thermophilus: characterization of the sulfhydryl state by protein-film voltammetry.

A disulfide bond, adjacent to the [2Fe-2S] cluster, is conserved in all high-potential Rieske proteins from the respiratory and photosynthetic cytochrome bc(1) and b(6)f complexes but is absent from the low-potential, bacterial dioxygenase Rieske proteins. The role of the disulfide is unclear, since cysteine mutants have resulted in only apoprotein. The high stability of the soluble Thermus thermophilus Rieske protein permits chemical reduction of the disulfide bond and characterization of the sulfhydryl (dithiol) form by protein-film voltammetry. The effect of disulfide reduction on the cluster potential is small (DeltaE(0)' <or= -0.04 V) and attributed to relaxation of the disulfide tether between the protein loops ligating the cluster, including possible mechanical strain release and hydrogen-bonding modification. Above pH 6 an additional decrease in potential of the sulfhydryl form is assigned to the nearby negatively charged thiolates (DeltaE(0)' -0.16 to -0.12 V); the histidine-ligand nitrogen pKs are correspondingly increased. Entropies of reduction for the native and dithiolate forms are equal (-48 +/- 5 J K(-1) mol(-1), pH 7-8); thus changes in reduction potential are enthalpic in origin. Following sulfhydryl alkylation the cluster redox properties mirror those of the native protein (DeltaE(0)' approximately -0.1 V) over all pHs. While a sustained electrode potential of -0.85 V fails to reduce the disulfide, the free sulfhydryls recombine upon an oxidative excursion, at low pH, to restore the native redox properties. This unique behavior is attributed to preorganization of the two thiolate groups upon uptake of one or more protons by the sulfhydryl pair.