Abstract
o-Phenanthroline and m-phenanthroline both inhibit the electron transfer activity of lauryl maltoside-solubilized yeast bc1 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 c1 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 Fe2+. Surprisingly, ferroin, the o-phenanthroline-ferrous sulfate complex, also inhibits the bc1 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 bc1 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 bc1 activity is reduced to 50%. In contrast to the solubilized form, the bc1 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 bc1 complex under different conditions and should provide a sensitive tool for determination of the relative stability of mutant enzymes.
Highlights
Doreductase, or bc1 complex, transfers electrons from ubiquinol to cytochrome c [1]
Inactivation of the bc1 Complex Using o- and m-Phenanthroline—We have found that when OP is added to lauryl maltoside-solubilized mitochondria, it acts as an inactivator of the bc1 complex
We have shown that OP and MP both inhibit the electron transfer activity of the yeast bc1 complex
Summary
(Received for publication, December 26, 1996, and in revised form, March 27, 1997). Hans Boumans‡, Monique C. Ferroin can only inhibit electron transfer activity by about 50% This behavior is discussed in relation to the dimeric structure of the bc complex, and we conclude that ferroin binds to only one of the two protomers. This enzyme complex may consist of up to 11 subunits, as in the bovine heart complex [2], but only three subunits contain prosthetic groups and are directly involved in electron transfer These subunits are cytochrome b, cytochrome c1, and the Rieske Fe-S protein and they form the “minimal,” or “core” complex. The OP-ferrous sulfate complex (ferroin) is found to have an inhibitory effect on the bc complex but of a different type The characteristics of this latter type of inhibition are discussed in relation to the dimeric structure of the bc complex.
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