Modulation of the Midpoint Potential of the [2Fe−2S] Rieske Iron Sulfur Center by Qo Occupants in the bc 1 Complex

• Published in: Biochemistry (Easton) Vol. 41; no. 48; pp. 14372 - 14382
• Main Authors: Shinkarev, Vladimir P, Kolling, Derrick R. J, Miller, Tim J, Crofts, Antony R
• Format: Journal Article
• Language: English
• Published: American Chemical Society 03-12-2002
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi026198c

Following addition of myxothiazol to antimycin-treated chromatophores from Rhodobacter sphaeroides poised at an ambient redox potential (E h) of ∼300 mV, the amplitude of the flash-induced cytochrome c 1 oxidation in the ms range increased, indicating a decrease in the availability of electrons from the immediate donor to c 1, the Rieske iron−sulfur protein (ISP). Because the effect was seen only over the limited E h range, we conclude that it is due to a decrease in the apparent midpoint redox potential (E m) of the ISP by about 40 mV on addition of myxothiazol. This is in line with the change in E m previously seen in direct redox titrations. Our results show that the reduced ISP binds with quinone at the Qo site with a higher affinity than does the oxidized ISP. The displacement of ubiquinone by myxothiazol leads to elimination of this preferential binding of the ISP reduced form and results in a shift in the midpoint potential of ISP to a more negative value. A simple hypothesis to explain this effect is that myxothiazol prevents formation of hydrogen bond of ubiquinone with the reduced ISP. We conclude that all Qo site occupants (ubiquinone, UHDBT, stigmatellin) that form hydrogen bonds with the reduced ISP shift the apparent E m of the ISP in the same direction to more positive values. Inhibitors that bind in the domain of the Qo site proximal to heme b L (myxothiazol, MOA−stilbene) and displace ubiquinone from the site cause a decrease in E m of ISP. We present a new formalism for treatment of the relation between E m change and the binding constants involved, which simplifies analysis. Using this formalism, we estimated that binding free energies for hydrogen bond formation with the Qo site occupant, range from the largest value of ∼23 kJ mol-1 in the presence of stigmatellin (appropriate for the buried hydrogen bond shown by structures), to a value of ∼3.5 kJ mol-1 in the native complex. We discuss this range of values in the context of a model in which the native structure constrains the interaction of ISP with the Qo site occupant so as to favor dissociation and the faster kinetics of unbinding necessary for rapid turnover.