ALTERATION OF THE MIDPOINT POTENTIAL AND CATALYTIC ACTIVITY OF THE RIESKE IRON-SULFUR PROTEIN BY CHANGES OF AMINO-ACIDS FORMING HYDROGEN-BONDS TO THE IRON-SULFUR CLUSTER

The crystal structure of the bovine Rieske iron-sulfur protein indicat es a sulfur atom (S-1) of the iron-sulfur cluster and the sulfur atom (S gamma of a cysteine residue that coordinates one of the iron atoms form hydrogen bonds with the hydroxyl groups of Ser-163 and Tyr-165, r espectively, We have altered the equivalent Ser-183 and Tyr-185 in the Saccharomyces cerevisiae Rieske iron-sulfur protein by site-directed mutagenesis of the iron-sulfur protein gene to examine how these hydro gen bonds affect the midpoint potential of the iron-sulfur cluster and how changes in the midpoint potential affect the activity of the enzy me, Eliminating the hydrogen bond from the hydroxyl group of Ser-183 t o S-1 of the cluster lowers the midpoint potential of the cluster by 1 30 mV, and eliminating the hydrogen bond from the hydroxyl group of Ty r-185 to S-gamma of Cys-159 lowers the midpoint potential by 65 mV, El iminating both hydrogen bonds has an approximately additive effect, lo wering the midpoint potential by 180 mV, Thus, these hydrogen bonds co ntribute significantly to the positive midpoint potential of the clust er but are not essential for its assembly, The activity of the bc(1) c omplex decreases with the decrease in midpoint potential, confirming t hat oxidation of ubiquinol by the iron-sulfur protein is the rate-limi ting partial reaction in the bc(1) complex, and that the rate of this reaction is extensively influenced by the midpoint potential of the ir on-sulfur cluster.