The cytochrome b subunit (subunit I) of the ubiquinolcytochrome c reductase (bc1 complex) is thought to participate in the formation of two quinone/quinol reaction centers, an oxidizing center (Qo) and a reducing center, in accordance with the quinone cycle mechanism. Threonine 160 is a highly conserved residue in a segment of subunit I that was shown to bind quinone and is placed near the putative Qo site in current models of the bc1 complex. Rhodobacter sphaeroides cells expressing bc1 complexes with Ser or Tyr substituted for Thr160 grow photosynthetically at a reduced rate, and cells expressing the mutated complexes produce an "elevated" level of the bc1 complex. The Ser substitution also affects the interaction of subunit IV with subunit I. Replacement of Thr160 by Ser results in about a 70% loss of the activity in the purified complex, whereas substitution by Tyr lowers the activity by more than 80%. Both replacements lower the apparent Km for ubiquinol. Electron paramagnetic resonance (EPR) spectroscopy shows that in the Ser substituted complex, the environments of the Rieske iron-sulfur cluster in subunit III and the high potential cytochrome b (b562) in subunit I have been modified. The spectra of the Ser160 and Tyr160 iron-sulfur clusters have become redox-insensitive, with a line shape resembling that of the native complex in the fully reduced state. The EPR signal of b562 in the Ser160 complex is shifted from g = 3.50 to g = 3.52, but otherwise the line shape is very similar to the spectrum of the native complex. Most of these results are consistent with current ideas regarding the structure and function of Qo in the bc1 complex, except for the alteration of the b562 EPR feature, because this heme is not thought to be located in proximity to Qo. Immunoblotting analysis showed that the Ser or Tyr substituted complex contained significantly less than a stoichiometric amount of subunit IV. The enzymatic activity of mutated bc1 complex was found to be activable by the addition of purified subunit IV. These results indicate that Thr160 plays an important role in the structure and/or function of the bc1 complex.