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 c(1) in the cytochrome bc(1) complex

We have changed nine conserved aromatic amino acids by site-directed mutage nesis of the cloned iron-sulfur protein gene to determine if any of these r esidues form an obligatory conduit for electron transfer within the iron-su lfur protein of the yeast cytochrome bc(1) complex. The residues include Wi ll, 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 prot eins, except for those containing a W152L and a W176L-F177L double mutation , for which the activity was similar to 45%. The crystal structures of the bc(1) complex indicate that F177 and H184 are at the surface of the iron-su lfur protein near the surface of cytochrome cr, but not directly in a linea r pathway between the iron-sulfur cluster and the cl heme. The pre-steady-s tate rates of reduction of cytochromes b and cl in mutants in which F177 an d H184 were changed to non-aromatic residues were approximately 70-85% of t he wildtype rates. There was a large decrease in iron-sulfur protein levels in mitochondrial membranes resulting from the W152L mutation and the W176- F177L double mutation, and a small decrease for the Y205L, W176L and F177L mutations. This indicates that the decreases in activity resulting from the se 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. (C) 1999 Elsev ier Science B.V. All rights reserved.