Roles of the disulfide bond and adjacent residues in determining the reduction potentials and stabilities of respiratory-type Rieske clusters

Rieske [2Fe-2S] clusters have reduction potentials which vary by over 500 mV, and which are pH dependent. In the cytochrome be, complex, the high-potential and low-pK values of the cluster may be important in the mechanism of quinol oxidation. Hydrogen bonds, from both side-chain and mainchain groups, are crucial for these properties, but solvent accessibility and a disulfide bond (present in only high-potential Rieske proteins) have been suggested to be important detertminants also. Previous studies have addressed the hydrogen bonds, disulfide bond, and a leucine residue which may restrict solvent access, by mutations in the cytochrome be, complex. However, influences on the complex (disruption of quinol binding and displacement of the Rieske domain) are difficult to deconvolute from intrinsic effects on the Rieske cluster. Here, the effects of similar mutations on cluster potential, pK values, and stability are characterized comprehensively in the isolated Rieske domain of the bovine protein. Hydrogen bonds from Ser163 and Tyr165 are important in increasing the reduction potential and decreasing the pK values. The disulfide has a limited effect on the redox properties, but is crucial for cluster stability, particularly in the oxidized state. Mutations of Leu142 had little effect on cluster potential, pK values, or stability, in contrast to the significant effects which were observed in the complex. The sum of the effects of all the mutated residues accounts for most of the differences between high- and low-potential Rieske proteins.