Elimination of the Disulfide Bridge in the Rieske Iron−Sulfur Protein Allows Assembly of the [2Fe-2S] Cluster into the Rieske Protein but Damages the Ubiquinol Oxidation Site in the Cytochrome bc₁ Complex

Abstract

The [2Fe-2S] cluster of the Rieske iron-sulfur protein is held between two loops of the protein that are connected by a disulfide bridge. We have replaced the two cysteines that form the disulfide bridge in the Rieske protein of Saccharomyces cerevisiae with tyrosine and leucine, and tyrosine and valine, to evaluate the effects of the disulfide bridge on assembly, stability, and thermodynamic properties of the Rieske iron-sulfur cluster. EPR spectra of the Rieske proteins lacking the disulfide bridge indicate the iron-sulfur cluster is assembled in the absence of the disulfide bridge, but there are significant shifts in all g values, indicating a change in the electronic structure of the [2Fe-2S] iron-sulfur center. In addition, the midpoint potential of the iron-sulfur cluster is lowered from 265 mV in the Rieske protein from wild-type yeast to 150 mV in the protein from the C164Y/C180L mutant and to 160 mV in the protein from the C164Y/C180V mutant. Ubiquinol-cytochrome c reductase activities of the bc(1) complexes with Rieske proteins lacking the disulfide bridge are less than 1% of the activity of the bc(1) complex from wild-type yeast, even though normal amounts of the iron-sulfur protein are present as judged by Western blot analysis. These activities are lower than the 105-115 mV decrease in the midpoint potential of the Rieske iron-sulfur cluster can account for. Pre-steady-state reduction of the bc(1) complexes with menadiol indicates that quinol is not oxidized through center P but is oxidized through center N. In addition, the levels of stigmatellin and UHDBT binding are markedly diminished, while antimycin binding is unaffected, in the bc(1) complexes with Rieske proteins lacking the disulfide bridge. Taken together, these results indicate that the ubiquinol oxidation site at center P is damaged in the bc(1) complexes with Rieske proteins lacking the disulfide bridge even though the iron-sulfur cluster is assembled into the Rieske protein.

The Small Subunit AroB of Arsenite Oxidase

“…3). Subsequent mutagenesis studies with mitochondrial Rieske proteins support a more moderate role for this disulfide bond with effects only on redox properties, catalytic activity, and the EPR spectrum (37,38). Our results suggest an even more modest role of this disulfide bond at least in Aro.…”

“…These residues have been implicated in the stability, the increase of E m value, and the catalytic activity of the protein (see Refs. [35][36][37][38]. In contrast, these Cys are absent in dioxygenase ferredoxins, a fact which had been considered to be a crucial parameter setting these latter centers apart from those of the Rieske-cyt b complexes and that gave rise to the name "Rieske-type" clusters.…”

“…None of the residues that replace the two Cys, i.e. Phe/Gly, in these natural cases have, however, been tested in mutagenesis studies (37,38).…”

“…Even if the effect of Rieske-cyt b inhibitors on Aro is greatly lower compared with Rieske-cyt b itself (i.e. 100%; see for example (37)), the fact that stigmatellin and quinols inhibit 35% of the Aro activity and perturb the EPR spectrum indicate that Aro binds quinols. Whether this interaction has a physiological role is beyond the scope of this study.…”

Role of a novel disulfide bridge within the all-beta fold of soluble Rieske proteins

“…Also, a disulfide bond has been shown to contribute significantly to the stability of the [2Fe-2S] plant-type ferredoxin from the hyperthermophilic bacterium Aquifex aeolicus, as its presence increases the melting temperature from 113 to 121°C [6]. One last example is given from the respiratory-type Rieske proteins, in which a strictly conserved disulfide in the immediate vicinity of the [2Fe-2S] cluster (within 5 Å ) stabilizes the cluster binding loops and modulates the redox potential [10].…”