SUBSTITUTIONS AT POSITION-146 OF CYTOCHROME-B AFFECT DRASTICALLY THE PROPERTIES OF HEME B(L) AND THE Q(O) SITE OF RHODOBACTER-CAPSULATUS CYTOCHROME BC(1) COMPLEX

The cytochrome (cyt) b subunit of ubihydroquinone: cytochrome c oxidor eductase (bc(1) complex) contains four invariant glycine (G) residues proposed to be essential for proper packing of the high and low potent ial (b(H) and b(L)) hemes of the bc(1) complex. One of these residues, G146 located in the transmembrane helix C of cyt b of Rhodobacter cap sulatus, was substituted with A and V using site-directed mutagenesis, and the effects of these substitutions on the properties of the ubiqu inone oxidation (Q(0)) site and heme b(L) of the bc(1) complex were an alyzed. The mutants G146A and V produced properly assembled but cataly tically defective bc(1) complexes that are unable to support photosynt hetic 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 l ight-activated single turnover rates were significantly lower than tho se of a wild-type complex. The dark potentiometric titrations revealed no significant changes in the redox midpoint potentials (E(m,7)) of t he high (b(H)) and low (b(L)) potential hemes of cyt b in both G146A a nd V mutants. However, EPR spectroscopy of the [2Fe-2S] cluster of the bc(1) complex indicated that the Q(0) site of the mutant enzymes were unoccupied. Moreover, the g(z) signal of heme b(L), but not that of h eme b(H), was modified both in G146A and V, suggesting that the geomet ry of its ligands has been distorted. These findings indicate that thi s region of cyt b must be well packed around heme b(L) since even a sl ight 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 b( L), alters substrate accessibility and binding to the Q(0) site, and r enders the bc(1) complex inactive.