Tyrosine 147 of cytochrome b is required for efficient electron transfer at the ubihydroquinone oxidase site (Q(o)) of the cytochrome bc(1) complex

In Rhodobacter capsulatus, tyrosine (Y) 147 is a highly conserved residue of the cyt b subunit of the bc(1) complex. It is located in the vicinity of residues altered in spontaneous inhibitor resistant mutants that affect the ubihydroquinone oxidase (Q(o)) site of this enzyme. In this work, Y147 was substituted with phenylalanine (F), valine (V), serine (S), and alanine (A) using site-directed mutagenesis in an effort to investigate its specific role in the Q(o) site. Of the four mutants obtained, Y147S and Y147A exhibited very low ubihydroquinone:cyt c reductase activities and were unable to support photosynthetic growth (Ps) while Y147F and Y147V were Ps(+). In all mutants, no changes in the redox midpoint potentials (E(m7)) of the cyt b(H) and cyt b(L), the occupancy of the Q(o) site by Q/QH(2), and the flash-induced reverse electron transfer kinetics from Q(i) to cyt b(H) were observed. On the other hand, rates of electron transfer from Q(o) to cyt b(H) were mildly reduced (2-3-fold) in Y147F and V but dramatically decreased (about 20-fold) in Y147A and S, localizing the defect to the Q(o) site. Thus, Y147A and S are members of a novel class of Q(o) site mutants that affect the Q(o) site catalysis without perturbing the accessibility or binding of the substrate. Additional insight to the role of Y147 on ubihydroquinone oxidation was gained by analyzing the Ps(+) revertants of these mutants. Two pseudorevertants contained a second mutation [isoleucine (I) or valine (V)] at the highly conserved M154 position, six residues away from Y147. These hydrophobic second-site substitutions restored electron transfer rates from Q(o) to cyt b(H) to near-wild type levels and conferred resistance to the Q(o) site inhibitor, myxothiazol. Considering that M154V mutation alone has no effect on the Q(o) site, the compensatory effects of the M154V or M154I substitutions are only needed when position 147 is occupied with a small amino acid side chain such as A or S. These spatial interactions between the positions 147 and 154 suggest that efficient electron transfer at the Q(o) site of the bet complex requires either bulky side chains at position 147 or hydrophobic side chains at position 154 when the former position is occupied with small residues. Thus, the overall packing and hydrophobicity of the Q(o) site appear crucial for maximum catalytic activity.