The [2Fe-2S] centre of the cytochrome bc complex in Bacillus firmus OF4 in EPR: an example of a menaquinol-oxidizing Rieske centre

The EPR and electrochemical properties of the Rieske [2Fe-2S] centre from the facultatively alkalophilic eubacterium Bacillus firmus strain OF4 are described. The redox midpoint potential of the Rieske centre was determined to be +105 ± 10 mV at pH 7.0. This value was found to be independent of pH up to pH 8 and to decrease at higher pH values with a slope slightly steeper than -60 mV/pH unit. With respect to its spectral characteristics the Rieske centre of B. firmus OF4 shows features typical for the [2Fe-2S] centres of cytochrome bc complexes. The alteration of the EPR lineshape between the ascorbate-and dithionite-reduced samples is indicative for an interaction of the Rieske centre with the menaquinone pool. The orientation of the g-tensor (the g_z and g_y axes are parallel to the membrane plane, g_x is oriented perpendicular) is similar to that from Rieske clusters from other cytochrome bc₁ and b₆f complexes. The Q_o-site inhibitors UHDBT and DBMIB affect the EPR spectrum of the Rieske centre. The effect of DBMIB is different from what has been reported for UQ/PQ-oxidizing Rieske centres. Furthermore, the binding of the Q_o-site inhibitor stigmatellin induces changes in the EPR lineshape, but has no influence on the redox midpoint potential of the cluster. The lack of a redox effect upon addition of stigmatellin and the influence of DBMIB on the EPR spectrum is proposed to be characteristic for Rieske centres from Gram-positive bacteria. The results obtained are compared to data sets from a variety of Rieske centres from different organisms. Together with previous results, these data support the definition of a new subset of Rieske centres with unique properties. Furthermore, the implications of the determined electrochemical parameters of the Rieske centre for the functioning of the electron transport chain of B. firmus under alkalophilic conditions are discussed.