Interplay between the hinge region of iron sulphur protein and the Qo site in the bc1 complex — Analysis of Plasmodium-like mutations in the yeast enzyme

Abstract

The respiratory chain bc1 complex is central to mitochondrial bioenergetics and the target of antiprotozoals. We characterized a modified yeast bc1 complex that more closely resemble Plasmodium falciparum enzyme. The mutant version was generated by replacing ten cytochrome b Qo site residues by P. falciparum equivalents. The Plasmodium-like changes caused a major dysfunction of the catalytic mechanism of the bc1 complex resulting in superoxide overproduction and respiratory growth defect. The defect was corrected by substitution of the conserved residue Y279 by a phenylalanine, or by mutations in or in the vicinity of the hinge domain of the iron-sulphur protein. It thus appears that side-reactions can be prevented by the substitution Y279F or the modification of the iron-sulphur protein hinge region. Interestingly, P. falciparum - and all the apicomplexan - contains an unusual hinge region. We replaced the yeast hinge region by the Plasmodium version and combined it with the Plasmodium-like version of the Qo site. This combination restored the respiratory growth competence. It could be suggested that, in the apicomplexan, the hinge region and the cytochrome b Qo site have co-evolved to maintain catalytic efficiency of the bc1 complex Qo site.

The antimalarial compound ELQ‐400 is an unusual inhibitor of the bc1 complex, targeting both Qo and Qi sites

“…None of the mutations had a severe effect on the catalytic site as K M and TN (turnover) values were only moderately changed (Data S1). The most affected mutants were PF‐Y279 and PF‐Y279S, as previously observed .…”

“…The ELQs are weaker inhibitors of the yeast enzyme than the stoichiometric inhibitor antimycin. In the same conditions, 50% inhibition was obtained with around 0.5 antimycin per bc 1 complex . Thus, the IC 50 value for antimycin is > 100‐fold lower than that for ELQ‐400.…”

“…PF‐Y279S combined the atovaquone resistance mutation Y279S (Y268S in P. falciparum ) with the 10 mutations of PF‐Y279. We previously showed that PF‐Y279S was markedly resistant to atovaquone, while PF‐Y279 was sensitive, and PF2 and PF8 displayed a moderate increase of resistance .…”

Human Mitochondrial Cytochrome b Variants Studied in Yeast: Not All Are Silent Polymorphisms

“…Using that approach, we have previously assessed the impact of human disease mutations on complex III and used mutants with severe respiratory function defect to explore possible compensation mechanisms [Fisher et al., , ; Meunier et al., ]. The yeast model is also an easy‐to‐use tool for characterizing acquired resistance mutations reported in mt‐cyb of pathogens after treatment by drugs targeting complex III or to explore the structural basis of the natural differential sensitivity of complex III to inhibitors [Hill et al., ; Song et al., , ].…”

Mutational analysis of the Qi-site proton pathway in yeast cytochrome bc1 complex

“…The difference between the reduction rate in the absence and the presence of added superoxide dismutase gives the contribution of the cytochrome c reduction by SO to the overall cytochrome c reductase activity. No SO production was detected (not shown), in contrast to the observed SO overproduction caused by Plasmodium-like mutations in the Q o -site [9] Thus there was no indication of a severe Q-cycle dysfunction in the PFQi11 and F225L-K228L bc 1 complex, in the conditions of the assays, explaining the respiratory growth defect.…”