Inhibitor effects on redox-linked protonations of the b haems of the mitochondrial bc1 complex

Abstract

The effects of pH and inhibitors on the spectra and redox properties of the haems b of the bc 1 complex of beef heart submitochondrial particles were investigated. The major findings were: (1) both haems have a weakly redox-linked protonatable group with p K ox and p K red of around 6 and 8; (2) at pH values above 7, haem b H becomes heterogeneous in its redox behaviour. This heterogeneity is removed by the Q i site inhibitors antimycin A, funiculosin and HQNO, but not by the Q o site inhibitors myxothiazol or stigmatellin; (3) of all inhibitors tested only funiculosin had a large effect on the E m 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 b H 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 b H and ubiquinone species bound at the Q i site. Simulations closely fit both the haem b H data and known semiquinone data only if it is assumed that semiquinone bound to oxidised haem b H is EPR-silent.