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Inhibitor effects on redox-linked protonations of the b haems of the mitochondrial bc1 complex
P R Rich1, A E Jeal, S A Madgwick
1Glynn Research Institute, Bodmin, U.K.
Insights
Investigating beef heart bc1 complex, researchers found pH and inhibitors affect haem b redox properties. Qi site inhibitors, but not Qo site inhibitors, resolve heterogeneous redox behavior in haem bH at higher pH.
Area of Science:
- Biochemistry
- Bioenergetics
- Mitochondrial Respiration
Background:
- The bc1 complex (cytochrome bc1) is crucial for cellular respiration, mediating electron transfer from ubiquinol to cytochrome c.
- Understanding the redox properties and proton linkage of its haem b centers is key to elucidating electron transfer mechanisms.
Purpose of the Study:
- To investigate the influence of pH and various inhibitors on the spectral and redox characteristics of haems b within the beef heart bc1 complex.
- To characterize the protonatable groups and redox-linked heterogeneity of haem bH.
Main Methods:
- Spectrophotometric analysis of haem redox potentials.
- Titration experiments under varying pH and anaerobic conditions.
- Application of site-specific inhibitors (antimycin A, funiculosin, HQNO, myxothiazol, stigmatellin).
- Computer simulations of redox behavior.
Main Results:
- Both haems exhibit weakly redox-linked protonatable groups (pKox ~6, pKred ~8).
- Haem bH shows pH-dependent heterogeneous redox behavior above pH 7, which is normalized by Qi site inhibitors (antimycin A, funiculosin, HQNO) but not Qo site inhibitors.
- Funiculosin uniquely altered the Em/pH profile of haem b.
- Spectral shifts upon inhibitor binding typically occurred at the haem closest to the binding site.
- Artefactual shifts in midpoint potentials were observed under aerobic titrations with cyanide.
- A model involving redox interaction between haem bH and Qi-site ubiquinone species explained the heterogeneous titration behavior.
Conclusions:
- The redox properties of haems b in the bc1 complex are sensitive to pH and specific inhibitor binding.
- Qi site inhibitors modulate the heterogeneous redox behavior of haem bH, suggesting a functional interaction with the ubiquinone binding site.
- Accurate redox measurements require strict anaerobic conditions to avoid artefacts.
Abstract:
The effects of pH and inhibitors on the spectra and redox properties of the haems b of the bc1 complex of beef heart submitochondrial particles were investigated. The major findings were: (1) both haems have a weakly redox-linked protonatable group with pKox and pKred of around 6 and 8; (2) at pH values above 7, haem bH becomes heterogeneous in its redox behaviour. This heterogeneity is removed by the Qi site inhibitors antimycin A, funiculosin and HQNO, but not by the Qo site inhibitors myxothiazol or stigmatellin; (3) of all inhibitors tested only funiculosin had a large effect on the Em/pH profile of either haem b. In all cases where definite effects were found, the haem most affected was that thought to be closest to the site of inhibitor binding; (4) spectral shifts of haem groups caused by inhibitor binding were usually, but not always, of the haem group closest to the binding site; (5) titrations with succinate/fumarate were in reasonable agreement with redox-mediated data provided that strict anaerobiosis was maintained. Apparent large shifts of haem midpoint potentials with antimycin A and myxothiazol could be produced in aerobic succinate/fumarate titrations in the presence of cyanide, as already reported in the literature, but these were artefactual; (6) the heterogeneous haem bH titration behaviour can be simulated with a model similar to that proposed by Salerno et al. (J. Biol. Chem. (1989) 264, 15398-15403) in which there is redox interaction between haem bH and ubiquinone species bound at the Qi site. Simulations closely fit both the haem bH data and known semiquinone data only if it is assumed that semiquinone bound to oxidised haem bH is EPR-silent.