Aromatic amino acids in the Rieske iron-sulfur protein do not form an obligatory conduit for electron transfer from the iron-sulfur cluster to the heme of cytochrome c1 in the cytochrome bc1 complex

We have changed nine conserved aromatic amino acids by site-directed mutagenesis of the cloned iron-sulfur protein gene to determine if any of these residues form an obligatory conduit for electron transfer within the iron-sulfur protein of the yeast cytochrome bc1 complex. The residues include W111, F117, W152, F173, W176, F177, H184, Y205 and F207. Greater than 70% of the catalytic activity was retained for all of the mutated iron-sulfur proteins, except for those containing a W152L and a W176L-F177L double mutation, for which the activity was approximately 45%. The crystal structures of the bc1 complex indicate that F177 and H184 are at the surface of the iron-sulfur protein near the surface of cytochrome c1, but not directly in a linear pathway between the iron-sulfur cluster and the c1 heme. The pre-steady-state rates of reduction of cytochromes b and c1 in mutants in which F177 and H184 were changed to non-aromatic residues were approximately 70-85% of the wild-type rates. There was a large decrease in iron-sulfur protein levels in mitochondrial membranes resulting from the W152L mutation and the W176L-F177L double mutation, and a small decrease for the Y205L, W176L and F177L mutations. This indicates that the decreases in activity resulting from these amino acid changes are due to instability of the altered proteins. These results show that these aromatic amino acids are unnecessary for electron transfer, but several are required for structural stability.