Differential Inhibition of the Yeast bc 1Complex by Phenanthrolines and Ferroin

o -Phenanthroline and m -phenanthroline both inhibit the electron transfer activity of lauryl maltoside-solubilized yeast bc 1 complex progressively with time. Pre-steady-state kinetics indicate that these compounds bind to the complex on the intermembrane space side, thereby blocking reduction of cytochrome b via the ubiquinol oxidation site. o -Phenanthroline is additionally capable of chelating an iron atom derived from the Rieske Fe-S cluster, thereby distorting the structure of the Rieske protein. EPR analysis shows that the secondary effect of o -phenanthroline occurs after initial inactivation and that m -phenanthroline, which lacks chelating activity, does not affect the Rieske Fe-S cluster. Spectral analysis shows that the b and c 1 cytochromes are still dithionite-reducible after inactivation by o -phenanthroline, indicating that they remain intact. Inactivation by o -phenanthroline can be prevented by the addition of Fe 2+ . Surprisingly, ferroin, the o -phenanthroline-ferrous sulfate complex, also inhibits the bc 1 complex activity. In contrast to o -phenanthroline, this effect is instantaneous. The two types of inhibition are clearly distinguishable by pre-steady-state reduction kinetics. Interestingly, ferroin can only inhibit electron transfer activity by about 50%. This behavior is discussed in relation to the dimeric structure of the bc 1 complex, and we conclude that ferroin binds to only one of the two protomers. The rate of inactivation by o -phenanthroline is dependent on the incubation temperature and can be quantitated in terms of the half-life for a certain temperature, the time at which the bc 1 activity is reduced to 50%. In contrast to the solubilized form, the bc 1 complex in intact mitochondria is insensitive to o -phenanthroline, suggesting that the inactivation rate by o -phenanthroline is dependent on accessibility of the complex to the agent. Reaction with o -phenanthroline is thus a useful technique for study of structural stability of the bc 1 complex under different conditions and should provide a sensitive tool for determination of the relative stability of mutant enzymes.