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Complete thermodynamic characterization of reduction and protonation of the bc(1)-type Rieske [2Fe-2S] center of Thermus thermophilus.

Rieske iron-sulfur (2Fe-2S) clusters play a central role in energy transduction by the quinone:cytochrome c oxidoreductases of the respiratory and photosynthetic chains (the bc1 and b6f complexes) and in the bacterial degradation of aromatic compounds. 1 Distinguished from “ferredoxin-type” 2Fe-2S clusters by reduction potentials up to 700 mV higher, Rieske centers have one iron atom coordinated by two nitrogenous (histidine, His) ligands rather than two thiolate (cysteine, Cys) ligands. 2,3 While the high, pH-dependent potentials of bc1-type clusters are crucial for redox-coupled proton transfer, and for “bifurcation” of electron transfer within thebc1-complex, 4 bacterial dioxygenase-type Rieske clusters, with the same 2Fe-2S Rieske core, display lower and pH-independent potentials. 5-8 Here we present the first complete thermodynamic description of a Rieske cluster, the soluble Rieske-fragment of thebc1-complex from Thermus thermophilus (Tt) .9 Using protein-film cyclic voltammetry, 10 which allows rapid transfer of a sample of protein molecules between different solutions, we have measured reduction potentials over a wide range of pH (up to pH 14). We have thus determined p K values for the His ligands in both redox states, and the reduction potential of each protonation state. Interpretation of these data now reveals how the His ligands dictate the unique physical properties of Rieske clusters. Figure 1 shows how the reduction potential ([2Fe-2S] 2+/1+) of theTt Rieske cluster varies between pH 3 and 14. 11 By defining this curve in three different ionic strengths (0.01, 0.1, and 2 M NaCl) we immediately identified the effect of a distant, ionizable amino acid residue in the low pH region. Since this weakly coupled residue can be “decoupled” by high salt concentrations, we do not consider it important to the intrinsic properties of the cluster and will therefore discuss only data recorded at the highest ionic strength. 12 The higher-pH data are uncomplicated by this distant charge, although E and pK values vary, as expected, with ionic-strength. 13 Figure 1 also shows fits to the data obtained using the thermodynamic scheme, Scheme 1, and eq 1 derived from it.14