The cytochrome (cyt) b subunit of ubihydroquinone: cytochrome c oxidoreductase (bc1 complex) contains four invariant glycine (G) residues proposed to be essential for proper packing of the high and low potential (bH and bL) hemes of the bc1 complex. One of these residues, G146 located in the transmembrane helix C of cyt b of Rhodobacter capsulatus, was substituted with A and V using site-directed mutagenesis, and the effects of these substitutions on the properties of the ubiquinone oxidation (Qo) site and heme bL of the bc1 complex were analyzed. The mutants G146A and V produced properly assembled but catalytically defective bc1 complexes that are unable to support photosynthetic growth. The steady-state ubihydroquinone: cytochrome c reductase activities of the mutant complexes were about one-tenth of that of a parental strain overproducing the wild-type enzyme. Similarly, their light-activated single turnover rates were significantly lower than those of a wild-type complex. The dark potentiometric titrations revealed no significant changes in the redox midpoint potentials (Em.7) of the high (bH) and low (bL) potential hemes of cyt b in both G146A and V mutants. However, EPR spectroscopy of the [2Fe-2S] cluster of the bc1 complex indicated that the Qo site of the mutant enzymes were unoccupied. Moreover, the gz signal of heme bL, but not that of heme bH, was modified both in G146A and V, suggesting that the geometry of its ligands has been distorted. These findings indicate that this region of cyt b must be well packed around heme bL since even a slight increase in the size of the amino acid side chain at position 146 (such as G to A) greatly perturbs the spatial conformation of heme bL, alters substrate accessibility and binding to the Qo site, and renders the bc1 complex inactive.