Analysis of inhibitor binding to the mitochondrial cytochrome c reductase by fluorescence quench titration

Abstract

The binding characteristics of inhibitors of the mitochondrial cytochrome c reductase were studied by fluorescence quench titration. Based on the standard binding equation, the applied numerical method allowed the online recorded titration curves to be interpreted by fitting the K d , the number of binding sites, and the specific fluorescence of the free and the bound inhibitor. For the Q i , center, 2‐n‐nonyl‐4‐hydroxyquinoline N ‐oxide and for the Q o center ( E )‐β‐methoxyacrylate‐stilbene (MOA‐stilbene) were used as fluorescing inhibitors. The experiments could be extended to other, non‐fluorescing inhibitors by competition analysis. Using this method we were able to compare the binding behaviour of Q i and Q o center inhibitors under different redox states of the enzyme using the same experimental set up. We studied the competition between inhibitors of the cytochrome c reductase representative for all subgroups and demonstrated that at least three inhibitor binding sites exist, two located in the Q o center, one located in the Q i center. Determination of the dissociation constants of the oxidized, the partially reduced and the fully reduced enzyme showed that inhibitor binding at the Q i center is not redox‐dependent. In contrast, the binding of MOA‐stilbene to the Q o center is decreased after reduction of the iron‐sulfur center and cytochrome c 1 whereas this redox change increases the affinity for a Q o center inhibitor of the hydroxynaphthoquinone type, 3‐n‐undecyl‐2‐hydroxy‐naphthoquinone. From these results, aware of the fact that the inhibitory mechanism at the Q o center is a non‐competitive one, we made the hypothesis of a ‘catalytic switch’ to explain both the bifurcation of electron flow and the inhibition at the Q o center. A steric blockage of one of two conformational states could serve as a cogent explanation for the great structural variability of the inhibitors and differential effects on the redox centers exerted by the inhibitors. Moreover, the proposed ‘switch’ gives some insight into other experimental results which are difficult to explain with the ubiquinone cycle as currently formulated.